Genomic organization of mouse and human sGC

ABSTRACT

Murine cDNA encoding the alpha 1 subunit of soluble guanylyl cyclase (sGC) and additional sequence to the known 3′ noncoding part of beta1 subunit of soluble guanylyl cyclase are identified herein. The new genes are further used for expression of encoded proteins. The new part of the beta 1  cDNA sequences is further used for screening of regulatory factors associated with modulation of the expression of the beta 1  sGC subunit.

[0001] The present application claims the benefit of U.S. Provisional Application Serial No. 60/233,500 filed on Sept. 19, 2000, the entire text of which is herein incorporated by reference.

[0002] The government owns rights in the present invention pursuant to grant provided by the John S. Dunn Foundation, the Harold and Leila Y. Mathers Foundation and the University of Texas.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates generally to the fields of genomic characterization. More particularly, it concerns nucleic acids and proteinaceous sequence of soluble guanylyl cyclase, and assays for compounds that affect its function in NO-dependent signal transduction.

[0005] 2. Description of Related Art

[0006] NO-dependent signal transduction is associated with a number of important physiological processes, including smooth muscle relaxation (Huang, 2000), platelet aggregation (Severina, 1998), neurotransmission (Dawson et al., 1998), cellular differentiation (Boss, 1989) and apoptotic cell death (Thippeswamy, 1997; Li et al., 1997; Liu, 1999). Soluble guanylyl cyclase (sGC), a NO-stimulated hemoprotein, which converts guanosine triphosphate to cyclic guanosine monophosphate, is a key element in these processes.

[0007] Soluble guanylyl cyclase is a heterodimer consisting of α and β subunits (Kamisaki et al., 1986), which are encoded by separate genes (Nakane and Murad, 1994). A heme prosthetic group is crucial for the stimulation of the enzyme by NO (Garbers, 1979; Ignarro et al. 1982). The enzyme has been purified from various animal tissues (Garbers, 1979; Gerzer et al., 1981; Ohlstein et al., 1982) and corresponding cDNAs were cloned from various vertebrate species, including rat (Nakane et al., 1988; Nakane et al., 1990), human (Giuili et al., 1992), bovine (Koesling et al., 1988; Koesling et al., 1990) and fish (Mikami et al., 1998). At least two isoforms for each subunit of the enzyme have been identified in various species, prompting a recent revision of the nomenclature of sGC subunits (Zabel et al., 1998). Although isoforms for both subunits were detected at the MRNA level in various tissues and were found to have an overlapping tissue distribution, until recently only the α₁/β₁ heterodimeric enzyme has been isolated from native sources. However, Russwurm and co-workers described an additional α₂/β₁heterodimer, which was shown previously to be catalytically active in vitro (Russwurm et al., 1998). Alternatively spliced transcripts for both human α (Riutter et al., 2000) and β (Behrends et al., 2000) subunits have also been reported. mRNA for the human α₁subunit undergoes alternative splicing, resulting in several mRNA species that are N-terminally truncated. The α₂₁subunit, an alternatively spliced variant of α₂, has been detected in several mammalian cell lines and tissues at the MRNA level Behrends et al., 1995), and a β₁cDNA splice variant has been detected in humans (Chhajlani et al., 1991).

[0008] Evidence that sGC activity is regulated both at the protein and MRNA levels has begun to emerge. Several groups have reported that such treatments as forskolin, dibutyryl-cAMP or 3-isobutyl-1-methyl xanthine (Papapetropoulos et al., 1995), endotoxin and/or IL-1β (Papapetropoulos et al., 1996), NO-donating compounds (Filippov et al., 1997) and nerve growth factor (Liu et al., 1997) affect the sGC mRNA levels in various cell types. There is also evidence that levels of sGC mRNA expression are subject to developmental regulation (Bloch et al., 1997).

[0009] Despite the significant role sGC plays in numerous physiological processes, little is known about the genomic organization of the genes for this enzyme in mammalian species. Recently, the genomic organization for the α₁and β₁subunits of sGC in Medaka fish (Mikami et al., 1999) and the β-subunit of sGC in mosquito (Anopheles gambiae) were reported (Caccone et al., 1999). Tandem genomic organization and evidence of directly coordinated transcription for α₁and β₁subunits in Medaka fish indicate the possibility of a similar mechanism of expression of sGC subunts in all vertebrates. Co-expression of both α and β subunits in transfected cells is required for enzyme activity (Nakane et al., 1990). Furthermore, the α₁and β₁sGC genes have been localized to the same chromosome in rat and human (Giuili et al., 1993). Co-expression of both α₁and β₁ subunits is obligatory for enzyme activity in rat lung (8;13) and human cerebral cortex, cerebellum and lung (Zabel et al., 1998). However, the ratio of expression levels for both subunits varies in a tissue dependent manner (Zabel et al., 1998), indicating that the regulation of expression for these subunits is not tightly coordinated as was indicated for Medic fish.

[0010] Despite an increasing interest in genetic aspects of sac regulation, relatively little is known about the genes or the promoter regions of mammalian GCS. There remains a need for nucleic and proteinaceous composition of sGC to identify agents that regulate its function, as well as allow the production of animal models with increased or decreased sGC function.

SUMMARY OF THE INVENTION

[0011] The present invention overcomes the deficiencies of the art by providing nucleic acids encoding the alpha1 subunit of soluble guanylyl cyclase (sGC) and additional sequence to the known 3′ noncoding part of beta1 subunit of soluble guanylyl cyclase. The present invention also provides sGC protein and nucleic acid compositions, screening assays for modulators of sGC expression and activity, and animal models comprising sGC with altered expression or activity.

[0012] Unless otherwise specified, as used herein, “sGC” may refer to nucleic acids encoding alpha1 subunit and/or beta1 subunit of sGC, or proteinaceous compositions encoded by such nucleic acids.

[0013] The invention first provides an isolated nucleic acid comprising a region having a nucleotide sequence that encodes the polypeptide sequences of SEQ ID NO: 2. In certain embodiments, the region is further defined as having the nucleotide sequence of the nucleotide sequence of SEQ ID NO: 1.

[0014] The invention provides an isolated and purified polynucleotide comprising a base sequence that is identical or complementary to a segment of at least 15 contiguous bases of SEQ ID NO: 1. In certain embodiments the polynucleotide hybridizes to a polynucleotide that a polypeptide comprising the amino acid residue sequence of SEQ ID NO: 1 or to the complement of such a sequence. In certain embodiments the polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 20 contiguous bases of SEQ ID NO: 1. In certain embodiments the polynucleotide comprises a base sequence that is identical or complementary to a insegment of at least 25 contiguous bases of SEQ ID NO: 2. In certain embodiments the polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 30 contiguous bases of SEQ ID NO: 1. In certain embodiments the polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 35 contiguous bases of SEQ ID NO: 1. In certain embodiments the polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 50 contiguous bases of SEQ ID NO: 1. In certain embodiments the polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 75 contiguous bases of SEQ ID NO: 1. In certain embodiments the polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 100 contiguous bases of SEQ ID NO: 1. In certain embodiments the polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 150 contiguous bases of SEQ ID NO: 1. In certain embodiments the polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 200 contiguous bases of SEQ ID NO: 1. In certain embodiments the polynucleotide comprises a base sequence that is identical or complementary to all contiguous bases of SEQ ID NO: 1.

[0015] The invention provides an expression vector comprising a polynucleotide that encodes a polypeptide comprising an amino acid residue sequence of SEQ ID NO: 2. In certain embodiments the polynucleotide comprises the nucleotide base sequence of SEQ ID NO: 1. In certain embodiments the polynucleotide is operatively linked to an enhancer-promoter.

[0016] The invention provides a recombinant host cell comprising a polynucleotide that encodes a polypeptide comprising an amino acid residue sequence of SEQ ID NO: 2. In certain embodiments the host cell comprising a polynucleotide that encodes a polypeptide comprising the amino acid residue sequence of SEQ ID NO: 2. In certain embodiments the polynucleotide comprises the nucleotide base sequence of SEQ ID NO: 1. In certain embodiments the polynucleotide is introduced into the cell by transformation of the cell with a vector comprising the polynucleotide. In certain embodiments the host cell expresses the polynucleotide to produce the polypeptide. In certain embodiments the cell is a PC12 cell, a CHO cell or a COS cell. In certain embodiments the cell is an E. coli cell. In certain embodiments the cell is a yeast cell.

[0017] The invention provides a process for preparing a cell expressing a polypeptide comprising the steps of: transfecting a cell with a polynucleotide that encodes a polypeptide comprising an amino acid residue sequence of SEQ ID NO: 2 to produce a transformed host cell; and maintaining the transformed host cell under biological conditions sufficient for expression of the polypeptide in the host cell. In certain embodiments the polynucleotide comprises a region having a nucleotide sequence of SEQ ID NO: 1. In certain embodiments the process is further defined as a process for preparing a cell expressing a polypeptide comprising the amino acid residue sequence of SEQ ID NO: 2. In certain embodiments the process further comprising purifying an expressed polypeptide having the amino acid sequence of SEQ ID NO: 2 from the transformed host cell.

[0018] The invention provides an isolated nucleic acid comprising a region having a nucleotide sequence of SEQ ID NO: 3.

[0019] The invention provides an isolated and purified polynucleotide comprising a base sequence that is identical or complementary to a segment of at least 15 contiguous bases of SEQ ID NO: 3. In certain embodiments the polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 20 contiguous bases of SEQ ID NO: 3. In certain embodiments the polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 25 contiguous bases of SEQ ID NO: 3. In certain embodiments the polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 30 contiguous bases of SEQ ID NO: 3. In certain embodiments the polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 35 contiguous bases of SEQ ID NO: 3. In certain embodiments the polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 50 contiguous bases of SEQ ID NO: 3. In certain embodiments the polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 75 contiguous bases of SEQ ID NO: 3. In certain embodiments the polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 100 contiguous bases of SEQ ID NO: 3. In certain embodiments the polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 150 contiguous bases of SEQ ID NO: 3. In certain embodiments the polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 200 contiguous bases of SEQ ID NO: 3. In certain embodiments the polynucleotide comprises a base sequence that is identical or complementary to all contiguous bases of SEQ ID NO: 3.

[0020] The invention provides an expression vector comprising a polynucleotide that comprises the nucleotide base sequence of SEQ ID NO: 3. In certain embodiments the polynucleotide is operatively linked to an enhancer-promoter.

[0021] The invention provides recombinant host cell comprising a polynucleotide that comprises the nucleotide base sequence of SEQ ID NO: 3. In certain embodiments the polynucleotide is introduced into the cell by transformation of the cell with a vector comprising the polynucleotide. In certain embodiments the cell is a PC12 cell, a CHO cell or a COS cell. In certain embodiments the cell is an E. coli cell. In certain embodiments the cell is a yeast cell.

[0022] The invention provides an isolated nucleic acid molecule comprising a region having a nucleic acid sequence of SEQ ID NO: 4 or a fragment thereof, the region further defined as encoding a murine alpha1 soluble guanylyl cyclase possessing a genomic organization as shown in Table 6.

[0023] The invention provides an isolated nucleic acid molecule comprising a region having a nucleic acid sequence of SEQ ID NO: 5 or a fragment thereof, the region further defined as encoding a murine beta1 soluble guanylyl cyclase possessing a genomic organization as shown in Table 6.

[0024] The invention provides an isolated nucleic acid molecule comprising a region having a nucleic acid sequence of SEQ ID NO: 6 or a fragment thereof, the region further defined as encoding a murine alpha1 soluble guanylyl cyclase possessing a genomic organization as shown in Table 6.

[0025] The invention provides a method of detecting a nucleic acid comprising a region having a sequence of, or complementary to, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a portion of any of these. In certain embodiments the method is further defined as a method utilizing a hybridization technique. In certain embodiments the method is further defined as a method utilizing an amplification technique. In certain embodiments the method is further defined as a method utilizing Southern hybridization. In certain embodiments the method is further defined as a method utilizing Northern hybridization. In certain embodiments the method is further defined as a method utilizing PCR amplification. In certain embodiments the method is further defined as a method utilizing DNA microarray analysis.

[0026] The invention provides a method of analyzing protein-nucleic acid interactions utilizing a nucleic acid comprising a region having a sequence of, or complementary to, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a portion of any of these.

[0027] In certain embodiments the method is further defined as a method of analysis of DNA-protein interactions. In certain embodiments the method is defined as a method of RNA-protein interactions. In certain embodiments the method is further defined as a method of analysis of DNA-protein interactions. In certain embodiments the method is further defined as a method of analysis of RNA-protein interactions.

[0028] The invention provides a method of analyzing substance-nucleic acid interactions utilizing a nucleic acid comprising a region having a sequence of, or complementary to, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a portion of any of these. In certain embodiments the method is further defined as a method of screening for drugs. In certain embodiments the method is further defined as a method of drug development. In certain embodiments the method is further defined as a diagnostic method.

[0029] The invention provides a method of producing a transgenic animal utilizing a nucleic acid comprising a region having a sequence of, or complementary to, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a portion of any of these.

[0030] The invention provides an expression vector comprising a nucleic acid having region having a sequence of, or complementary to, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a portion of any of these. In certain embodiments the polynucleotide is operatively linked to an enhancer-promoter.

[0031] The invention provides a recombinant host cell comprising nucleic acid having region having a sequence of, or complementary to, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a portion of any of these. In certain embodiments the polynucleotide comprises the nucleotide base sequence of SEQ ID NO: 1. In certain embodiments the polynucleotide is introduced into the cell by transformation of the cell with a vector comprising the polynucleotide. In certain embodiments the host cell expresses the polynucleotide to produce the polypeptide. In certain embodiments the cell is a PC12 cell, a CHO cell or a COS cell. In certain embodiments the cell is an E. coli cell. In certain embodiments the cell is a yeast cell.

[0032] The invention provides a process for preparing a cell expressing a polypeptide comprising the steps of: transfecting a cell with a nucleic acid having region having a sequence of, or complementary to, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a portion of any of these to produce a transformed host cell; and maintaining the transformed host cell under biological conditions sufficient for expression of the polypeptide in the host cell. In certain embodiments the polynucleotide comprises a region having a nucleotide sequence of SEQ ID NO: 1. In certain embodiments the process is further defined as a process for preparing a cell expressing a polypeptide comprising the amino acid residue sequence of SEQ ID NO: 2. In certain embodiments the process further comprises purifying an expressed polypeptide from the transformed host cell. In certain embodiments the process is further defined a process of producing an active enzyme. In certain embodiments the active enzyme is employed in biochemical characterization, studies of drug-enzyme interactions, drug discovery, drug development, and/or design.

[0033] The invention provides a method for the detection of genetic and/or inherited and/or acquired human diseases utilizing a nucleic acid comprising a region having a sequence of, or complementary to, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a portion of any of these.

[0034] In certain embodiments the method is further defined as a method for detection of chromosomal abnormalities connected with cancer, hypertension, heart failure, stroke, neurodegenerative diseases, Alzheimers disease, Parkinsons disease, endocrinopathy, an inflammatory disorder, shock, sepsis, abnormal gasrointestinal motility, altered muscle disorder, altered movement disorder, ocular disorder, sensory disorder, or dermatological disorder. In certain embodiments the method is further defined as a method for the detection of point mutations, deletions, and/or insertions. In certain embodiments the method is further defined as a method for the detection of aberrations in splicing.

[0035] The invention provides a diagnostic kit for the detection of genetic and/or inherited and/or acquired human diseases utilizing a nucleic acid comprising a region having a sequence of, or complementary to, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a portion of any of these.

[0036] In certain embodiments the kit is further defined as a kit for detection of chromosomal abnormalities connected with cancer, hypertension, heart failure, stroke, neurodegenerative diseases, Alzheimers disease, Parkinsons disease, endocrinopathy, an inflammatory disorder, shock, sepsis, abnormal gasrointestinal motility, altered muscle disorder, altered movement disorder, ocular disorder, sensory disorder, or dermatological disorder. In certain embodiments the kit is further defined as a kit for the detection of point mutations, deletions, and/or insertions. In certain embodiments the kit is further defined as a kit for the detection of aberrations in splicing.

[0037] The invention provides a method of treating disease comprising utilizing a nucleic acid comprising a region having a sequence of, or complementary to, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a portion of any of these. In certain embodiments the method is further defined as a method of gene therapy. In certain embodiments the method is further defined as a method for treating chromosomal abnormalities connected with cancer, hypertension, heart failure, stroke, neurodegenerative diseases, Alzheimers disease, Parkinsons disease, endocrinopathy, an inflammatory disorder, shock, sepsis, abnormal gasrointestinal motility, altered muscle disorder, altered movement disorder, ocular disorder, sensory disorder, or dermatological disorder.

[0038] The invention provides a method of screening for drugs, drug design, and/or drug development comprising utilizing a nucleic acid comprising a region having a sequence of, or complementary to, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a portion of any of these.

[0039] Product sGC.

[0040] Product sGC for use as a medicament.

[0041] Use of sGC for the manufacture or a medicament for the treatment of disease, including but not limited to cancer, hypertension, heart failure, stroke, neurodegenerative diseases, Alzheimers disease, Parkinsons disease, endocrinopathy, an inflammatory disorder, shock, sepsis, abnormal gasrointestinal motility, altered muscle disorder, altered movement disorder, ocular disorder, sensory disorder, or dermatological disorder.

[0042] As used herein, “any range derivable therein” means a range selected from the numbers described in the specification, and “any integer derivable therein” means any integer between such a range.

[0043] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising” , the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more.

[0044] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0045] The cDNA cloning, chromosomal localization and structure of the mouse genes for the α₁and β₁subunits of sGC and a comparative analyses with the human sGC genes, using the Human Genome database (NCBI) is provided herein. Organizational and regulatory sequences for human and mouse alpha1 and beta1 soluble guanylyl cyclase genes were characterized and the chromosomal localization were identified. The regulatory sequences are further used in the screening for the structure and identity of regulatory factors and compounds for the modulation of the expression of sGC genes and in studies of associated physiological or pathological pathways. Hormonal regulation of the MRNA levels for α₁and β₁sGC subunits has been identified. The DNA information on the genomic organization and loci of the genes are further used for the diagnosis of genetic abnormalities (polymorpisms and mutations) in sGC genes and association with a potential predisposition to cardiovascular, neurological, genetic, inherited and other diseases for the purpose of diagnosis and treatment.

[0046] I. sGC Nucleic Acids

[0047] In one embodiment, the present invention discloses a novel nucleic acid sequence and a novel protein encoded by the nucleic acid that has homology to the sGC family of genes and proteins.

[0048] A. Genes and DNA Segments

[0049] Important aspects of the present invention concern isolated DNA segments and recombinant vectors encoding sGC proteins, polypeptides or peptides, and the creation and use of recombinant host cells through the application of DNA technology, that express a wild-type, polymorphic or mutant sGC, using the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6, and biologically functional equivalents thereof.

[0050] The present invention concerns DNA segments, isolatable from mammalian cells, such as mouse or human cells, that are free from total genomic DNA and that are capable of expressing a protein, polypeptide or peptide that has sGC activity. As used herein, the term “DNA segment” refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding sGC refers to a DNA segment that contains wild-type, polymorphic or mutant sGC coding sequences yet is isolated away from, or purified free from, total mammalian genomic DNA. Included within the term “DNA segment”, are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.

[0051] Similarly, a DNA segment comprising an isolated or purified sGC gene refers to a DNA segment including sGC protein, polypeptide or peptide coding sequences and, in certain aspects, regulatory sequences, isolated substantially away from other naturally occurring genes or protein encoding sequences. In this respect, the term “gene” is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, cDNA sequences and engineered segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins and mutants of sGC encoded sequences.

[0052] “Isolated substantially away from other coding sequences” means that the gene of interest, in this case the sGC gene, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.

[0053] In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that encode a sGC protein, polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in, SEQ ID NO: 2, corresponding to the sGC designated “murine sGC”.

[0054] The term “a sequence essentially as set forth in SEQ ID NO: 2” means that the sequence substantially corresponds to a portion of SEQ ID NO: 2 and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of SEQ ID NO: 2.

[0055] The term “biologically functional equivalent” is well understood in the art and is further defined in detail herein. Accordingly, sequences that have about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, and any range derivable therein, such as, for example, about 70% to about 80%, and more preferably about 81% and about 90%; or even more preferably, between about 91% and about 99%; of amino acids that are identical or functionally equivalent to the amino acids of SEQ ID NO: 2 will be sequences that are “essentially as set forth in SEQ ID NO: 2” , provided the biological activity of the protein is maintained. In particular embodiments, the biological activity of a sGC protein, polypeptide or peptide, or a biologically functional equivalent, comprises binding to one or more proteases, particularly sGC.

[0056] In certain other embodiments, the invention concerns isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6. The term “essentially as set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6” is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6 and has relatively few codons that are not identical, or functionally equivalent, to the codons of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6. Again, DNA segments that encode proteins, polypeptide or peptides exhibiting sGC activity will be most preferred.

[0057] The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine and serine, and also refers to codons that encode biologically equivalent amino acids. For optimization of expression of sGC in human cells, the codons are shown in Table 1 in preference of use from left to right. Thus, the most preferred codon for alanine is thus “GCC”, and the least is “GCG” (see Table 1 below). Codon usage for various organisms and organelles can be found at the website http://www.kazusa.orjp/codon/, incorporated herein by reference, allowing one of skill in the art to optimize codon usage for expression in various organisms using the disclosures herein. Thus, it is contemplated that codon usage may be optimized for other animals, as well as other organisms such as a prokaryote (e.g., an eubacteria, an archaea), an eukaryote (e.g., a protist, a plant, a fungi, an animal), a virus and the like, as well as organelles that contain nucleic acids, such as mitochondria or chloroplasts, based on the preferred codon usage as would be known to those of ordinary skill in the art. TABLE 1 Preferred Human DNA Codons Amino Acids Codons Alanine Ala A GCC GCT GCA GCG Cysteine Cys C TGC TGT Aspartic acid Asp D GAC GAT Glutamic acid Glu E GAG GAA Phenylalanine Phe F TTC TTT Glycine Gly G GGC GGG GGA GGT Histidine His H CAC CAT Isoleucine Ile I ATC ATT ATA Lysine Lys K AAG AAA Leucine Leu L CTG CTC TTG CTT CTA TTA Methionine Met M ATG Asparagine Asn N AAC AAT Proline Pro P CCC CCT CCA CCG Glutamine Gln Q CAG CAA Arginine Arg R CGC AGG CGG AGA CGA CGT Serine Ser S AGC TCC TCT AGT TCA TCG Threonine Thr T ACC ACA ACT ACG Valine Val V GTG GTC GTT GTA Tryptophan Trp W TGG Tyrosine Tyr Y TAC TAT

[0058] It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5′ or 3′ sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein, polypeptide or peptide activity where an amino acid sequence expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5′ or 3′ portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.

[0059] Excepting intronic or flanking regions, and allowing for the degeneracy of the genetic code, sequences that have about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, and any range derivable therein, such as, for example, about 70% to about 80%, and more preferably about 81% and about 90%; or even more preferably, between about 91% and about 99%; of nucleotides that are identical to the nucleotides of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6 will be sequences that are “essentially as set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6”.

[0060] B. Nucleic Acid Hybridization

[0061] The nucleic acid sequences disclosed herein also have a variety of uses, such as for example, utility as probes or primers in nucleic acid hybridization embodiments.

[0062] Naturally, the present invention also encompasses DNA segments that are complementary, or essentially complementary, to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6. Nucleic acid sequences that are “complementary” are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules. As used herein, the term “complementary sequences” means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6 under stringent conditions such as those described herein.

[0063] As used herein, “hybridization”, “hybridizes” or “capable of hybridizing” is understood to mean the forming of a double or triple stranded molecule or a molecule with partial double or triple stranded nature. The term “hybridization”, “hybridize(s)” or “capable of hybridizing” encompasses the terms “stringent condition(s)” or “high stringency” and the terms “low stringency” or “low stringency condition(s).”

[0064] As used herein “stringent condition(s)” or “high stringency” are those conditions that allow hybridization between or within one or more nucleic acid strand(s) containing complementary sequence(s), but precludes hybridization of random sequences. Stringent conditions tolerate little, if any, mismatch between a nucleic acid and a target strand. Such conditions are well known to those of ordinary skill in the art, and are preferred for applications requiring high selectivity. Non-limiting applications include isolating a nucleic acid, such as a gene or a nucleic acid segment thereof, or detecting at least one specific mRNA transcript or a nucleic acid segment thereof, and the like.

[0065] Stringent conditions may comprise low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50° C. to about 70° C. It is understood that the temperature and ionic strength of a desired stringency are determined in part by the length of the particular nucleic acid(s), the length and nucleotide base content of the target sequence(s), the charge composition of the nucleic acid(s), and to the presence or concentration of formamide, tetramethylammonium chloride or other solvent(s) in a hybridization mixture.

[0066] It is also understood that these ranges, compositions and conditions for hybridization are mentioned by way of non-limiting examples only, and that the desired stringency for a particular hybridization reaction is often determined empirically by comparison to one or more positive or negative controls. Depending on the application envisioned it is preferred to employ varying conditions of hybridization to achieve varying degrees of selectivity of a nucleic acid towards a target sequence. In a non-limiting example, identification or isolation of a related target nucleic acid that does not hybridize to a nucleic acid under stringent conditions may be achieved by hybridization at low temperature and/or high ionic strength. For example, a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37° C. to about 55° C. Under these conditions, hybridization may occur even though the sequences of probe and target strand are not perfectly complementary, but are mismatched at one or more positions. In another example, a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20° C. to about 55° C. Of course, it is within the skill of one in the art to further modify the low or high stringency conditions to suite a particular application. For example, in other embodiments, hybridization may be achieved under conditions of, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 1.0 mM dithiothreitol, at temperatures between approximately 20° C. to about 37° C. Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, at temperatures ranging from approximately 40° C. to about 72° C.

[0067] Accordingly, the nucleotide sequences of the disclosure may be used for their ability to selectively form duplex molecules with complementary stretches of genes or RNAs or to provide primers for amplification of DNA or RNA from tissues. Depending on the application envisioned, it is preferred to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence.

[0068] The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, enhancers, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

[0069] For example, nucleic acid fragments may be prepared that include a contiguous stretch of nucleotides identical to or complementary to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6, such as, for example, about 8, about 10 to about 14, or about 15 to about 20 nucleotides, and that are chromosome sized pieces, up to about 1,000,000, about 750,000, about 500,000, about 250,000, about 100,000, about 50,000, about 20,000, or about 10,000, or about 5,000 base pairs in length, with segments of about 3,000 being preferred in certain cases, as well as DNA segments with total lengths of about 1,000, about 500, about 200, about 100 and about 50 base pairs in length (including all intermediate lengths of these lengths listed above, i.e., any range derivable therein and any integer derivable therein such a range) are also contemplated to be useful.

[0070] For example, it will be readily understood that “intermediate lengths”, in these contexts, means any length between the quoted ranges, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, including all integers through the 200-500; 500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000; 5,000-10,000 ranges, up to and including sequences of about 12,001, 12,002, 13,001, 13,002, 15,000, 20,000 and the like.

[0071] Various nucleic acid segments may be designed based on a particular nucleic acid sequence, and may be of any length. By assigning numeric values to a sequence, for example, the first residue is 1, the second residue is 2, etc., an algorithm defining all nucleic acid segments can be created:

n to n+y

[0072] where n is an integer from 1 to the last number of the sequence and y is the length of the nucleic acid segment minus one, where n+y does not exceed the last number of the sequence. Thus, for a 10-mer, the nucleic acid segments correspond to bases 1 to 10, 2 to 11, 3 to 12 . . . and/or so on. For a 15-mer, the nucleic acid segments correspond to bases 1 to 15, 2 to 16, 3 to 17 . . . and/or so on. For a 20-mer, the nucleic segments correspond to bases 1 to 20, 2 to 21, 3 to 22 . . . and/or so on. In certain embodiments, the nucleic acid segment may be a probe or primer. As used herein, a “probe” generally refers to a nucleic acid used in a detection method or composition. As used herein, a “primer” generally refers to a nucleic acid used in an extension or amplification method or composition.

[0073] The use of a hybridization probe of between 17 and 100 nucleotides in length, or in some aspect of the invention even up to 1-2 Kb or more in length, allows the formation of a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than 20 bases in length are generally preferred, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of particular hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having stretches of 20 to 30 nucleotides, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production.

[0074] In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridization, as in PCR™, for detection of expression of corresponding genes, as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface to remove non-specifically bound probe molecules, hybridization is detected, or even quantified, by means of the label.

[0075] C. Nucleic Acid Amplification

[0076] Nucleic acid used as a template for amplification is isolated from cells contained in the biological sample, according to standard methodologies (Sambrook et al., 1989). The nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to convert the RNA to a complementary DNA. In one embodiment, the RNA is whole cell RNA and is used directly as the template for amplification.

[0077] Pairs of primers that selectively hybridize to nucleic acids corresponding to sGC genes are contacted with the isolated nucleic acid under conditions that permit selective hybridization. The term “primer”, as defined herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. Typically, primers are oligonucleotides from ten to twenty or thirty base pairs in length, but longer sequences can be employed. Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is preferred.

[0078] Once hybridized, the nucleic acid:primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as “cycles,” are conducted until a sufficient amount of amplification product is produced.

[0079] Next, the amplification product is detected. In certain applications, the detection may be performed by visual means. Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax technology).

[0080] A number of template dependent processes are available to amplify the marker sequences present in a given template sample. One of the best known amplification methods is the polymerase chain reaction (referred to as PCR™) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, each incorporated herein by reference in entirety.

[0081] Briefly, in PCR™, two primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence. An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase. If the marker sequence is present in a sample, the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated.

[0082] A reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of MRNA amplified. Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al., 1989. Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641, filed Dec. 21, 1990, incorporated herein by reference. Polymerase chain reaction methodologies are well known in the art.

[0083] Another method for amplification is the ligase chain reaction (“LCR”), disclosed in EPA No. 320 308, incorporated herein by reference in its entirety. In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCR™, bound ligated units dissociate from the target and then serve as “target sequences” for ligation of excess probe pairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence.

[0084] Qbeta Replicase, described in PCT Application No. PCT/US87/00880, incorporated herein by reference, may also be used as still another amplification method in the present invention. In this method, a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence that can then be detected.

[0085] An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention.

[0086] Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation. A similar method, called Repair Chain Reaction (RCR), involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used in SDA. Target specific sequences can also be detected using a cyclic probe reaction (CPR). In CPR, a probe having 3′ and 5′ sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample. Upon hybridization, the reaction is treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion. The original template is annealed to another cycling probe and the reaction is repeated.

[0087] Still another amplification methods described in GB Application No. 2 202 328, and in PCT Application No. PCT/US89/01025, each of which is incorporated herein by reference in its entirety, may be used in accordance with the present invention. In the former application, “modified” primers are used in a PCR-like, template- and enzyme-dependent synthesis. The primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latter application, an excess of labeled probes are added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.

[0088] Other nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Gingeras et al., PCT Application WO 88/10315, incorporated herein by reference). In NASBA, the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA. These amplification techniques involve annealing a primer which has target specific sequences. Following polymerization, DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization. The double-stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNA's are reverse transcribed into single stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate target specific sequences.

[0089] Davey et al., EPA No. 329 822 (incorporated herein by reference in its entirety) disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention. The ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA). The resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5′ to its homology to the template. This primer is then extended by DNA polymerase (exemplified by the large “Klenow” fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.

[0090] Miller et al., PCT Application WO 89/06700 (incorporated herein by reference in its entirety) disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA (“ssDNA”) followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts. Other amplification methods include “RACE” and “one-sided PCR” (Frohman, 1990, incorporated herein by reference).

[0091] Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting “di-oligonucleotide”, thereby amplifying the di-oligonucleotide, may also be used in the amplification step of the present invention.

[0092] D. Nucleic Acid Detection

[0093] In certain embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of being detected. In preferred embodiments, one may desire to employ a fluorescent label or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known that can be employed to provide a detection means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.

[0094] In embodiments wherein nucleic acids are amplified, it may be desirable to separate the amplification product from the template and the excess primer for the purpose of determining whether specific amplification has occurred. In one embodiment, amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods (Sambrook et al., 1989).

[0095] Alternatively, chromatographic techniques may be employed to effect separation. There are many kinds of chromatography which may be used in the present invention: adsorption, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography.

[0096] Amplification products must be visualized in order to confirm amplification of the marker sequences. One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light. Alternatively, if the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.

[0097] In one embodiment, visualization is achieved indirectly. Following separation of amplification products, a labeled, nucleic acid probe is brought into contact with the amplified marker sequence. The probe preferably is conjugated to a chromophore but may be radiolabeled. In another embodiment, the probe is conjugated to a binding partner, such as an antibody or biotin, and the other member of the binding pair carries a detectable moiety.

[0098] In one embodiment, detection is by Southern blotting and hybridization with a labeled probe. The techniques involved in Southern blotting are well known to those of skill in the art and can be found in many standard books on molecular protocols. See Sambrook et al., 1989. Briefly, amplification products are separated by gel electrophoresis. The gel is then contacted with a membrane, such as nitrocellulose, permitting transfer of the nucleic acid and non-covalent binding. Subsequently, the membrane is incubated with a chromophore-conjugated probe that is capable of hybridizing with a target amplification product. Detection is by exposure of the membrane to x-ray film or ion-emitting detection devices.

[0099] One example of the foregoing is described in U.S. Pat. No. 5,279,721, incorporated by reference herein, which discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids. The apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.

[0100] Other methods for genetic screening to accurately detect mutations in genomic DNA, cDNA or RNA samples may be employed, depending on the specific situation.

[0101] Historically, a number of different methods have been used to detect point mutations, including denaturing gradient gel electrophoresis (“DGGE”), restriction enzyme polymorphism analysis, chemical and enzymatic cleavage methods, and others. The more common procedures currently in use include direct sequencing of target regions amplified by PCR™ (see above) and single-strand conformation polymorphism analysis (“SSCP”).

[0102] Another method of screening for point mutations is based on RNase cleavage of base pair mismatches in RNA/DNA and RNA/RNA heteroduplexes. As used herein, the term “mismatch” is defined as a region of one or more unpaired or mispaired nucleotides in a double-stranded RNA/RNA, RNA/DNA or DNA/DNA molecule. This definition thus includes mismatches due to insertion/deletion mutations, as well as single and multiple base point mutations.

[0103] U.S. Pat. No. 4,946,773 describes an RNase A mismatch cleavage assay that involves annealing single-stranded DNA or RNA test samples to an RNA probe, and subsequent treatment of the nucleic acid duplexes with RNase A. After the RNase cleavage reaction, the RNase is inactivated by proteolytic digestion and organic extraction, and the cleavage products are denatured by heating and analyzed by electrophoresis on denaturing polyacrylamide gels. For the detection of mismatches, the single-stranded products of the RNase A treatment, electrophoretically separated according to size, are compared to similarly treated control duplexes. Samples containing smaller fragments (cleavage products) not seen in the control duplex are scored as positive.

[0104] Currently available RNase mismatch cleavage assays, including those performed according to U.S. Pat. No. 4,946,773, require the use of radiolabeled RNA probes. Myers and Maniatis in U.S. Pat. No. 4,946,773 describe the detection of base pair mismatches using RNase A. Other investigators have described the use of an E. coli enzyme, RNase I, in mismatch assays. Because it has broader cleavage specificity than RNase A, RNase I would be a desirable enzyme to employ in the detection of base pair mismatches if components can be found to decrease the extent of non-specific cleavage and increase the frequency of cleavage of mismatches. The use of RNase I for mismatch detection is described in literature from Promega Biotech. Promega markets a kit containing RNase I that is shown in their literature to cleave three out of four known mismatches, provided the enzyme level is sufficiently high.

[0105] The RNase protection assay was first used to detect and map the ends of specific mRNA targets in solution. The assay relies on being able to easily generate high specific activity radiolabeled RNA probes complementary to the MRNA of interest by in vitro transcription. Originally, the templates for in vitro transcription were recombinant plasmids containing bacteriophage promoters. The probes are mixed with total cellular RNA samples to permit hybridization to their complementary targets, then the mixture is treated with RNase to degrade excess unhybridized probe. Also, as originally intended, the RNase used is specific for single-stranded RNA, so that hybridized double-stranded probe is protected from degradation. After inactivation and removal of the RNase, the protected probe (which is proportional in amount to the amount of target MRNA that was present) is recovered and analyzed on a polyacrylamide gel.

[0106] The RNase Protection assay was adapted for detection of single base mutations. In this type of RNase A mismatch cleavage assay, radiolabeled RNA probes transcribed in vitro from wild-type sequences, are hybridized to complementary target regions derived from test samples. The test target generally comprises DNA (either genomic DNA or DNA amplified by cloning in plasmids or by PCR™), although RNA targets (endogenous mRNA) have occasionally been used. If single nucleotide (or greater) sequence differences occur between the hybridized probe and target, the resulting disruption in Watson-Crick hydrogen bonding at that position (“mismatch”) can be recognized and cleaved in some cases by single-strand specific ribonuclease. To date, RNase A has been used almost exclusively for cleavage of single-base mismatches, although RNase I has recently been shown as useful also for mismatch cleavage. There are recent descriptions of using the MutS protein and other DNA-repair enzymes for detection of single-base mismatches.

[0107] E. Cloning sGC Genes

[0108] The present invention contemplates cloning sGC genes or cDNAs from animal (e.g., mammalian) organisms. A technique often employed by those skilled in the art of protein production today is to obtain a so-called “recombinant” version of the protein, to express it in a recombinant cell and to obtain the protein, polypeptide or peptide from such cells. These techniques are based upon the “cloning” of a DNA molecule encoding the protein from a DNA library, i.e., on obtaining a specific DNA molecule distinct from other portions of DNA. This can be achieved by, for example, cloning a cDNA molecule, or cloning a genomic-like DNA molecule.

[0109] The first step in such cloning procedures is the screening of an appropriate DNA library, such as, for example, from a mouse, rat, monkey or human. The screening protocol may utilize nucleotide segments or probes that are designed to hybridize to cDNA or genomic sequences of sGCs. Additionally, antibodies designed to bind to the expressed sGC proteins, polypeptides, or peptides may be used as probes to screen an appropriate mammalian DNA expression library. Alternatively, activity assays may be employed. The operation of such screening protocols are well known to those of skill in the art and are described in detail in the scientific literature, for example, in Sambrook et al. (1989), incorporated herein by reference. Moreover, as the present invention encompasses the cloning of genomic segments as well as CDNA molecules, it is contemplated that suitable genomic cloning methods, as known to those in the art, may also be used.

[0110] As used herein “designed to hybridize” means a sequence selected for its likely ability to hybridize to a mammalian sGC gene, for example due to the expected high degree of homology between the human sGC gene and the sGC genes from other mammals. Also included are segments or probes altered to enhance their ability to hybridize to or bind to a mammalian sGC gene. Additionally, these regions of homology also include amino acid sequences of 4 or more consecutive amino acids selected and/or altered to increase conservation of the amino acid sequences in comparison to the same or similar region of residues in the same or related genes in one or more species. Such amino acid sequences may derived from amino acid sequences encoded by the sGC gene, and more particularly from the isolated sequences of SEQ ID NO: 2.

[0111] General methods for screening a mammalian DNA library are exemplified by, but not limited to, the methods detailed in Example 1 herein below. Nucleotide probes may derived from nucleotide sequences from the human sGC sequence, and more particularly from the isolated sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6. Such sequences may be used as probes for hybridization or oligonucleotide primers for PCR™. Designing such sequences may involve selection of regions of highly conserved nucleotide sequences between various species for a particular gene or related genes, relative to the general conservation of nucleotides of the gene or related genes in one or more species. Comparison of the amino acid sequences conserved between one or more species for a particular gene may also be used to determine a group of 4 or more consecutive amino acids that are conserved relative to the protein encoded by the gene or related genes. The nucleotide probe or primers may then be designed from the region of the gene that encodes the conserved sequence of amino acids.

[0112] One may also prepare fusion proteins, polypeptides and peptides, e.g., where the sGC proteinaceous material coding regions are aligned within the same expression unit with other proteins, polypeptides or peptides having desired functions, such as for purification or immunodetection purposes (e.g., proteinaceous compostions that may be purified by affinity chromatography and enzyme label coding regions, respectively).

[0113] Encompassed by the invention are DNA segments encoding relatively small peptides, such as, for example, peptides of from about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 35, about 40, about 45, to about 50 amino acids in length, and more preferably, of from about 15 to about 30 amino acids in length; as set forth in SEQ ID NO: 2 and also larger polypeptides up to and including proteins corresponding to the full-length sequences set forth in SEQ ID NO: 2, and any range derivable therein and any integer derivable therein such a range.

[0114] In addition to the “standard” DNA and RNA nucleotide bases, modified bases are also contemplated for use in particular applications of the present invention. A table of exemplary, but not limiting, modified bases is provided herein below. TABLE 2 Purine and Pyrmidine Derivatives or Analogs Modified base Modified base Abbr. description Abbr. description ac4c 4-acetylcytidine Mam5s2u 5-methoxyaminomethyl- 2-thiouridine chm5u 5-(carboxyhydroxyl- Man q Beta,D- methyl)uridine mannosylqueosine Cm 2′-O-methylcytidine Mcm5s2u 5-methoxycarbonyl- methyl-2-thiouridine Cmnm5s2u 5-carboxymethyl- Mcm5u 5-methoxycarbonyl- aminomethyl-2-thio- methyluridine ridine Cmnm5u 5-carboxymethyl- Mo5u 5-methoxyuridine aminomethyluridine D Dihydrouridine Ms2i6a 2-methylthio-N6- isopentenyladenosine Fm 2′-O-methylpseudo- Ms2t6a N-((9-beta-D- uridine ribofuranosyl-2- methylthiopurine-6- yl)carbamoyl)threonine gal q beta,D-galac- Mt6a N-((9-beta-D- tosylqueosine ribofuranosylpurine-6- yl)N-methyl- carbamoyl)threonine Gm 2′-O-methyl- Mv Uridine-5-oxyacetic acid guanosine methylester I Inosine o5u Uridine-5-oxyacetic acid (v) I6a N6-isopentenyl- Osyw Wybutoxosine adenosine m1a 1-methyladenosine P Pseudouridine m1f 1-methylpseudo- Q Queosine uridine m1g 1-methylguanosine s2c 2-thiocytidine m1I 1-methylinosine s2t 5-methyl-2-thiouridine m22g 2,2-dimethyl- s2u 2-thiouridine guanosine m2a 2-methyladenosine s4u 4-thiouridine m2g 2-methylguanosine T 5-methyluridine m3c 3-methylcytidine t6a N-((9-beta-D- ribofuranosylpurine-6- yl)carbamoyl)threonine m5c 5-methylcytidine Tm 2′-O-methyl-5-methyl- uridine m6a N6-methyladenosine Um 2′-O-methyluridine m7g 7-methylguanosine Yw Wybutosine Mam5u 5-methylamino- X 3-(3-amino-3- methyluridine carboxypropyl)uridine, (acp3)u

[0115] II. Mutagenesis, Peptidomimetics and Rational Drug Design

[0116] It will also be understood that this invention is not limited to the particular nucleic acid and amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID 5 NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6. Recombinant vectors and isolated DNA segments may therefore variously include these coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include such coding regions or may encode biologically functional equivalent proteins, polypeptides or peptides that have variant amino acids sequences.

[0117] The DNA segments of the present invention encompass biologically functional equivalent sGC proteins, polypeptides, and peptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteinaceous compositions thus encoded. Alternatively, functionally equivalent proteins, polypeptides or peptides may be created via the application of recombinant DNA technology, in which changes in the protein, polypeptide or peptide structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced, for example, through the application of site-directed mutagenesis techniques as discussed herein below, e.g., to introduce improvements to the antigenicity of the proteinaceous composition or to test mutants in order to examine sGC activity at the molecular level.

[0118] Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins, polypeptides or peptides, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.

[0119] In general, the technique of site-specific mutagenesis is well known in the art. As will be appreciated, the technique typically employs a bacteriophage vector that exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.

[0120] In general, site-directed mutagenesis is performed by first obtaining a single-stranded vector, or melting of two strands of a double stranded vector which includes within its sequence a DNA sequence encoding the desired proteinaceous molecule. An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared. This primer is then annealed with the single-stranded DNA preparation, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.

[0121] The preparation of sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of genes may be obtained. For example, recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.

[0122] As modifications and changes may be made in the structure of the sGC genes, nucleic acids (e.g., nucleic acid segments) and proteinaceous molecules of the present invention, and still obtain molecules having like or otherwise desirable characteristics, such biologically functional equivalents are also encompassed within the present invention.

[0123] For example, certain amino acids may be substituted for other amino acids in a proteinaceous structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies, binding sites on substrate molecules or receptors, or such like. Since it is the interactive capacity and nature of a proteinaceous molecule that defines that proteinaceous molecule's biological functional activity, certain amino acid sequence substitutions can be made in a proteinaceous molecule sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a proteinaceous molecule with like (agonistic) properties. It is thus contemplated that various changes may be made in the sequence of sGC proteins, polypeptides or peptides, or the underlying nucleic acids, without appreciable loss of their biological utility or activity.

[0124] Equally, the same considerations may be employed to create a protein, polypeptide or peptide with countervailing, e.g., antagonistic properties. This is relevant to the present invention in which sGC mutants or analogues may be generated. For example, a sGC mutant may be generated and tested for sGC activity to identify those residues important for sGC activity. sGC mutants may also be synthesized to reflect a sGC mutant that occurs in the human population and that is linked to the development of cancer. Such mutant proteinaceous molecules are particularly contemplated for use in generating mutant-specific antibodies and such mutant DNA segments may be used as mutant-specific probes and primers.

[0125] While discussion has focused on functionally equivalent polypeptides arising from amino acid changes, it will be appreciated that these changes may be effected by alteration of the encoding DNA; taking into consideration also that the genetic code is degenerate and that two or more codons may code for the same amino acid. A table of amino acids and their codons is presented herein above for use in such embodiments, as well as for other uses, such as in the design of probes and primers and the like.

[0126] In terms of functional equivalents, it is well understood by the skilled artisan that, inherent in the definition of a “biologically functional equivalent” protein, polypeptide, peptide, gene or nucleic acid, is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule and still result in a molecule with an acceptable level of equivalent biological activity. Biologically functional equivalent peptides are thus defined herein as those peptides in which certain, not most or all, of the amino acids may be substituted.

[0127] In particular, where shorter length peptides are concerned, it is contemplated that fewer amino acids changes should be made within the given peptide. Longer domains may have an intermediate number of changes. The full length protein will have the most tolerance for a larger number of changes. Of course, a plurality of distinct proteins/polypeptide/peptides with different substitutions may easily be made and used in accordance with the invention.

[0128] It is also well understood that where certain residues are shown to be particularly important to the biological or structural properties of a protein, polypeptide or peptide, e.g., residues in binding regions or active sites, such residues may not generally be exchanged. In this manner, functional equivalents are defined herein as those peptides which maintain a substantial amount of their native biological activity.

[0129] Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all a similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents.

[0130] To effect more quantitative changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

[0131] The importance of the hydropathic amino acid index in conferring interactive biological function on a proteinaceous molecule is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0132] It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biological functional equivalent protein, polypeptide or peptide thereby created is intended for use in immunological embodiments, as in certain embodiments of the present invention. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a proteinaceous molecule, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the proteinaceous molecule.

[0133] As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

[0134] In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0135] In addition to the sGC peptidyl compounds described herein, it is contemplated that other sterically similar compounds may be formulated to mimic the key portions of the peptide structure. Such compounds, which may be termed peptidomimetics, may be used in the same manner as the peptides of the invention and hence are also functional equivalents.

[0136] Certain mimetics that mimic elements of proteinaceous molecule's secondary structure are described in Johnson et al. (1993). The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteinaceous molecules exists chiefly to orientate amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen. A peptide mimetic is thus designed to permit molecular interactions similar to the natural molecule.

[0137] Some successful applications of the peptide mimetic concept have focused on mimetics of β-turns within proteinaceous molecules, which are known to be highly antigenic. Likely β-turn structure within a polypeptide can be predicted by computer-based algorithms, as discussed herein. Once the component amino acids of the turn are determined, mimetics can be constructed to achieve a similar spatial orientation of the essential elements of the amino acid side chains.

[0138] The generation of further structural equivalents or mimetics may be achieved by the techniques of modeling and chemical design known to those of skill in the art. The art of receptor modeling is now well known, and by such methods a chemical that binds sGC can be designed and then synthesized. It will be understood that all such sterically designed constructs fall within the scope of the present invention.

[0139] In addition to the 20 “standard” amino acids provided through the genetic code, modified or unusual amino acids are also contemplated for use in the present invention. A table of exemplary, but not limiting, modified or unusual amino acids is provided herein below. TABLE 3 Modified and Unusual Amino Acids Abbr. Amino Acid Abbr. Amino Acid Aad 2-Aminoadipic acid EtAsn N-Ethylasparagine Baad 3-Aminoadipic acid Hyl Hydroxylysine Bala Beta-alanine, beta-Amino- aHyl Allo-Hydroxylysine propionic acid Abu 2-Aminobutyric acid 3Hyp 3-Hydroxyproline 4Abu 4-Aminobutyric acid, piperidinic 4Hyp 4-Hydroxyproline acid Acp 6-Aminocaproic acid Ide Isodesmosine Ahe 2-Aminoheptanoic acid aIle Allo-Isoleucine Aib 2-Aminoisobutyric acid MeGly N-Methylglycine, sarcosine Baib 3-Aminoisobutyric acid MeIle N-Methylisoleucine Apm 2-Aminopimelic acid MeLys 6-N-Methyllysine Dbu 2,4-Diaminobutyric acid MeVal N-Methylvaline Des Desmosine Nva Norvaline Dpm 2,2′-Diaminopimelic acid Nle Norleucine Dpr 2,3-Diaminopropionic acid Orn Ornithine EtGly N-Ethylglycine

[0140] In one aspect, an compound may be designed by rational drug design to function as a sGC in inhibition of sGC. The goal of rational drug design is to produce structural analogs of biologically active compounds. By creating such analogs, it is possible to fashion drugs which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules. In one approach, one would generate a three-dimensional structure for the sGC protein of the invention or a fragment thereof. This could be accomplished by X-ray crystallography, computer modeling or by a combination of both approaches. An alternative approach, involves the random replacement of functional groups throughout the sGC protein, polypeptides or peptides, and the resulting affect on function determined.

[0141] It also is possible to isolate a sGC protein, polypeptide or peptide specific antibody, selected by a functional assay, and then solve its crystal structure. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of anti-idiotype would be expected to be an analog of the original antigen. The anti-idiotype could then be used to identify and isolate peptides from banks of chemically- or biologically-produced peptides. Selected peptides would then serve as the pharmacore. Anti-idiotypes may be generated using the methods described herein for producing antibodies, using an antibody as the antigen.

[0142] Thus, one may design drugs which have enhanced and improved, or reduced, biological activity, for example, NO-dependent signal transduction, relative to a starting sGC proteinaceous sequences. By virtue of the ability to recombinatly produce sufficient amounts of the sGC proteins, polypeptides or peptides, crystallographic studies may be preformed to determine the most likely sites for mutagenesis and chemical mimicry. In addition, knowledge of the chemical characteristics of these compounds permits computer employed predictions of structure-function relationships. Computer models of various polypeptide and peptide structures are also available in the literature or computer databases. In a non-limiting example, the Entrez database (http://www.ncbi.nlm.nih.gov/Entrez/) may be used by one of ordinary skill in the art to identify target sequences and regions for mutagenesis.

[0143] III. Recombinant Vectors, Host Cells and Expression

[0144] Recombinant vectors form an important further aspect of the present invention. The term “expression vector or construct” means any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The transcript may be translated into a proteinaceous molecule, but it need not be. Thus, in certain embodiments, expression includes both transcription of a gene and translation of a RNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid, for example, to generate antisense constructs.

[0145] Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA segment, whether encoding a full length protein or smaller polypeptide or peptide, is positioned under the transcriptional control of a promoter. A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrases “operatively positioned”, “under control” or “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.

[0146] The promoter may be in the form of the promoter that is naturally associated with an sGC gene, as may be obtained by isolating the 5′ non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCR technology, in connection with the compositions disclosed herein (PCR™ technology is disclosed in U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195, each incorporated herein by reference).

[0147] In other embodiments, it is contemplated that certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with an sGC gene in its natural environment. Such promoters may include promoters normally associated with other genes, and/or promoters isolated from any other bacterial, viral, eukaryotic, protist, or mammalian cell, and/or promoters made by the hand of man that are not “naturally occurring”, i.e., containing difference elements from different promoters, or mutations that increase, decrease, or alter expression.

[0148] Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type, organism, or even animal, chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al. (1989), incorporated herein by reference. The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins, polypeptides or peptides.

[0149] At least one module in a promoter generally functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.

[0150] Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase promoter, the spacing between promoter elements can be increased to 50 basepairs apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.

[0151] The particular promoter that is employed to control the expression of a nucleic acid is not believed to be critical, so long as it is capable of expressing the nucleic acid in the targeted cell. Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a human or viral promoter.

[0152] In various other embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter and the Rous sarcoma virus long terminal repeat can be used to obtain high-level expression of the instant nucleic acids. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression are contemplated as well, provided that the levels of expression are sufficient for a given purpose. Tables 4 and 5 below list several elements/promoters which may be employed, in the context of the present invention, to regulate the expression of an sGC gene. This list is not intended to be exhaustive of all the possible elements involved in the promotion of expression but, merely, to be exemplary thereof.

[0153] Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA. This ability to act over a large distance had little precedent in classic studies of prokaryotic transcriptional regulation. Subsequent work showed that regions of DNA with enhancer activity are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins.

[0154] The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.

[0155] Additionally any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB, http://www.epd.isb-sib.ch/) could also be used to drive expression. Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct. TABLE 4 Promoter and Enhancer Elements Promoter/Enhancer References Immunoglobulin Heavy Chain Banerji et al., 1983; Gilles et al., 1983; Grosschedl and Baltimore, 1985; Atchinson and Perry, 1986, 1987; Imler et al., 1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton et al.; 1990 Immunoglobulin Light Chain Queen and Baltimore, 1983; Picard and Schaffner, 1984 T-Cell Receptor Luria et al., 1987; Winoto and Baltimore, 1989; Redondo et al.; 1990 HLA DQ a and DQ β Sullivan and Peterlin, 1987 β-Interferon Goodbourn et al., 1986; Fujita et al., 1987; Goodbourn and Maniatis, 1988 Interleukin-2 Greene et al., 1989 Interleukin-2 Receptor Greene et al., 1989; Lin et al., 1990 MHC Class II 5 Koch et al., 1989 MHC Class II HLA-Dra Sherman et al., 1989 β-Actin Kawamoto et al., 1988; Ng et al.; 1989 Muscle Creatine Kinase Jaynes et al., 1988; Horlick and Benfield, 1989; Johnson et al., 1989 Prealbumin (Transthyretin) Costa et al., 1988 Elastase I Ornitz et al., 1987 Metallothionein Karin et al., 1987; Culotta and Hamer, 1989 Collagenase Pinkert et al., 1987; Angel et al., 1987 Albumin Gene Pinkert et al., 1987; Tronche et al., 1989, 1990 α-Fetoprotein Godbout et al., 1988; Campere and Tilghman, 1989 t-Globin Bodine and Ley, 1987; Perez- Stable and Constantini, 1990 β-Globin Trudel and Constantini, 1987 e-fos c-HA-ras Deschamps et al., 1985 Insulin Edlund et al., 1985 Neural Cell Adhesion Molecule Hirsh et al., 1990 (NCAM) α_(1-Antitrypain) Latimer et al., 1990 H2B (TH2B) Histone Hwang et al., 1990 Mouse or Type I Collagen Ripe et al., 1989 Glucose-Regulated Proteins Chang et al., 1989 (GRP94 and GRP78) Rat Growth Hormone Larsen et al., 1986 Human Serum Amyloid A (SAA) Edbrooke et al., 1989 Troponin I (TN I) Yutzey et al., 1989 Platelet-Derived Growth Factor Pech et al., 1989 Duchenne Muscular Dystrophy Klamut et al., 1990 SV40 Banerji et al., 1981; Moreau et al., 1981; Sleigh and Lockett, 1985; Firak and Subramanian, 1986; Herr and Clarke, 1986; Imbra and Karin, 1986; Kadesch and Berg, 1986; Wang and Calame, 1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al., 1988 Polyoma Swartzendruber and Lehman, 1975; Vasseur et al., 1980; Katinka et al., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983; de Villiers et al., 1984; Hen et al., 1986; Satake et al., 1988; Campbell and Villarreal, 1988 Retroviruses Kriegler and Botchan, 1982, 1983; Levinson et al., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze et al., 1986; Miksicek et al., 1986; Celander and Haseltine, 1987; Thiesen et al., 1988; Celander et al., 1988; Choi et al., 1988; Reisman and Rotter, 1989 Papilloma Virus Campo et al., 1983; Lusky et al., 1983; Spandidos and Wilkie, 1983; Spalholz et al., 1985; Lusky and Botchan, 1986; Cripe et al., 1987; Gloss et al., 1987; Hirochika et al., 1987; Stephens and Hentschel, 1987 Hepatitis B Virus Bulla and Siddiqui, 1986; Jameel and Siddiqui, 1986; Shaul and Ben-Levy, 1987; Spandau and Lee, 1988; Vannice and Levinson, 1988 Human Immunodeficiency Virus Muesing et al., 1987; Hauber and Cullan, 1988; Jakobovits et al., 1988; Feng and Holland, 1988; Takebe et al., 1988; Rosen et al., 1988; Berkhout et al., 1989; Laspia et al., 1989; Sharp and Marciniak, 1989; Braddock et al., 1989 Cytomegalovirus Weber et al., 1984; Boshart et al., 1985; Foecking and Hofstetter, 1986 Gibbon Ape Leukemia Virus Holbrook et al., 1987; Quinn et al., 1989

[0156] TABLE 5 Inducible Elements Element Inducer References MT II Phorbol Ester (TFA) Palmiter et al., 1982; Heavy metals Haslinger and Karin, 1985; Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin et al., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV (mouse Glucocorticoids Huang et al., 1981; mammary tumor Lee et al., 1981; Majors and virus) Varmus, 1983; Chandler et al., 1983; Lee et al., 1984; Ponta et al., 1985; Sakai et al., 1988 β-Interferon Poly(rI)x Tavernier et al., 1983 Poly(rc) Adenovirus 5 E2 Ela Imperiale and Nevins, 1984 Collagenase Phorbol Ester (TPA) Angel et al., 1987a Stromelysin Phorbol Ester (TPA) Angel et al., 1987b SV40 Phorbol Ester (TPA) Angel et al., 1987b Murine MX Gene Interferon, Newcastle Disease Virus GRP78 Gene A23187 Resendez et al., 1988 α-2-Macroglobu- IL-6 Kunz et al., 1989 lin Vimentin Serum Rittling et al., 1989 MHC Class I Interferon Blanar et al., 1989 Gene H-2κb HSP70 Ela, SV40 Large T Taylor et al., 1989; Taylor and Antigen Kingston, 1990a, b Proliferin Phorbol Ester-TPA Mordacq and Linzer, 1989 Tumor Necrosis FMA Hensel et al., 1989 Factor Thyroid Stimu- Thyroid Hormone Chatterjee et al., 1989 lating Hormone a Gene

[0157] Turning to the expression of the sGC proteinaceous molecules of the present invention, once a suitable clone or clones have been obtained, whether they be cDNA based or genomic, one may proceed to prepare an expression system. The engineering of DNA segment(s) for expression in a prokaryotic or eukaryotic system may be performed by techniques generally known to those of skill in recombinant expression. It is believed that virtually any expression system may be employed in the expression of the proteinaceous molecules of the present invention.

[0158] Both cDNA and genomic sequences are suitable for eukaryotic expression, as the host cell will generally process the genomic transcripts to yield functional mRNA for translation into proteinaceous molecules. Generally speaking, it may be more convenient to employ as the recombinant gene a cDNA version of the gene. It is believed that the use of a CDNA version will provide advantages in that the size of the gene will generally be much smaller and more readily employed to transfect the targeted cell than will a genomic gene, which will typically be up to an order of magnitude or more larger than the cDNA gene. However, it is contemplated that a genomic version of a particular gene may be employed where desired.

[0159] In expression, one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed. Preferred embodiments include the SV40 polyadenylation signal and the bovine growth hormone polyadenylation signal, convenient and known to function well in various target cells. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

[0160] The term “antisense nucleic acid” is intended to refer to the oligonucleotides complementary to the base sequences of DNA and RNA. Antisense oligonucleotides, when introduced into a target cell, specifically bind to their target nucleic acid and interfere with transcription, RNA processing, transport and/or translation. Targeting double-stranded (ds) DNA with oligonucleotide leads to triple-helix formation; targeting RNA will lead to double-helix formation.

[0161] Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. Antisense RNA constructs, or DNA encoding such antisense RNAs, may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject. Nucleic acid sequences comprising “complementary nucleotides” are those which are capable of base-pairing according to the standard Watson-Crick complementary rules. That is, that the larger purines will base pair with the smaller pyrimidines to form only combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T), in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA.

[0162] As used herein, the terms “complementary” or “antisense sequences” mean nucleic acid sequences that are substantially complementary over their entire length and have very few base mismatches. For example, nucleic acid sequences of fifteen bases in length may be termed complementary when they have a complementary nucleotide at thirteen or fourteen positions with only single or double mismatches. Naturally, nucleic acid sequences which are “completely complementary” will be nucleic acid sequences which are entirely complementary throughout their entire length and have no base mismatches.

[0163] While all or part of the gene sequence may be employed in the context of antisense construction, statistically, any sequence 17 bases long should occur only once in the human genome and, therefore, suffice to specify a unique target sequence. Although shorter oligomers are easier to make and increase in vivo accessibility, numerous other factors are involved in determining the specificity of hybridization. Both binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length. It is contemplated that oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more base pairs will be used. One can readily determine whether a given antisense nucleic acid is effective at targeting of the corresponding host cell gene simply by testing the constructs in vitro to determine whether the endogenous gene's function is affected or whether the expression of related genes having complementary sequences is affected.

[0164] In certain embodiments, one may wish to employ antisense constructs which include other elements, for example, those which include C-5 propyne pyrimidines. Oligonucleotides which contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression (Wagner et al., 1993).

[0165] As an alternative to targeted antisense delivery, targeted ribozymes may be used. The term “ribozyme” refers to an RNA-based enzyme capable of targeting and cleaving particular base sequences in oncogene DNA and RNA. Ribozymes either can be targeted directly to cells, in the form of RNA oligo-nucleotides incorporating ribozyme sequences, or introduced into the cell as an expression construct encoding the desired ribozymal RNA. Ribozymes may be used and applied in much the same way as described for antisense nucleic acids.

[0166] A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon and adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be “in-frame” with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.

[0167] It is proposed that sGC proteins, polypeptides or peptides may be co-expressed with other selected proteinaceous molecules, wherein the proteinaceous molecules may be co-expressed in the same cell or sGC gene may be provided to a cell that already has another selected proteinaceous molecule. Co-expression may be achieved by co-transfecting the cell with two distinct recombinant vectors, each bearing a copy of either of the respective DNA. Alternatively, a single recombinant vector may be constructed to include the coding regions for both of the proteinaceous molecules, which could then be expressed in cells transfected with the single vector. In either event, the term “co-expression” herein refers to the expression of both the sGC gene and the other selected proteinaceous molecules in the same recombinant cell.

[0168] As used herein, the terms “engineered” and “recombinant” cells or host cells are intended to refer to a cell into which an exogenous DNA segment or gene, such as a cDNA or gene encoding a sGC protein, polypeptide or peptide has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced exogenous DNA segment or gene. Engineered cells are thus cells having a gene or genes introduced through the hand of man. Recombinant cells include those having an introduced cDNA or genomic gene, and also include genes positioned adjacent to a promoter not naturally associated with the particular introduced gene.

[0169] To express a recombinant sGC protein, polypeptide or peptide, whether mutant or wild-type, in accordance with the present invention one would prepare an expression vector that comprises a wild-type, or mutant sGC proteinaceous molecule-encoding nucleic acid under the control of one or more promoters. To bring a coding sequence “under the control of” a promoter, one positions the 5′ end of the transcription initiation site of the transcriptional reading frame generally between about 1 and about 50 nucleotides “downstream” of (i.e., 3′ of) the chosen promoter. The ” upstream” promoter stimulates transcription of the DNA and promotes expression of the encoded recombinant protein, polypeptide or peptide. This is the meaning of “recombinant expression” in this context.

[0170] Many standard techniques are available to construct expression vectors containing the appropriate nucleic acids and transcriptional/translational control sequences in order to achieve protein, polypeptide or peptide expression in a variety of host-expression systems. Cell types available for expression include, but are not limited to, bacteria, such as E. coli and B. subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors.

[0171] Certain examples of prokaryotic hosts are E. coli strain RR1, E. coli LE392, E. coli B, E. coli X 1776 (ATCC No. 31537) as well as E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325); bacilli such as Bacillus subtilis; and other enterobacteriaceae such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species.

[0172] In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is often transformed using derivatives of pBR322, a plasmid derived from an E. coli species. pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of its own proteins.

[0173] In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, the phage lambda GEM™-11 may be utilized in making a recombinant phage vector which can be used to transform host cells, such as E. coli LE392.

[0174] Further useful vectors include pIN vectors (Inouye et al., 1985); and pGEX vectors, for use in generating glutathione S-transferase (GST) soluble fusion proteins for later purification and separation or cleavage. Other suitable fusion proteins are those with β-galactosidase, ubiquitin, and the like.

[0175] Promoters that are most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose and tryptophan (trp) promoter systems. While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling those of skill in the art to ligate them functionally with plasmid vectors.

[0176] The following details concerning recombinant protein production in bacterial cells, such as E. coli, are provided by way of exemplary information on recombinant protein production in general, the adaptation of which to a particular recombinant expression system will be known to those of skill in the art.

[0177] Bacterial cells, for example, E. coli, containing the expression vector are grown in any of a number of suitable media, for example, LB. The expression of the recombinant proteinaceous molecule may be induced, e.g., by adding IPTG to the media or by switching incubation to a higher temperature. After culturing the bacteria for a further period, generally of between 2 and 24 hours, the cells are collected by centrifugation and washed to remove residual media.

[0178] The bacterial cells are then lysed, for example, by disruption in a cell homogenizer and centrifuged to separate the dense inclusion bodies and cell membranes from the soluble cell components. This centrifugation can be performed under conditions whereby the dense inclusion bodies are selectively enriched by incorporation of sugars, such as sucrose, into the buffer and centrifugation at a selective speed.

[0179] If the recombinant proteinaceous molecule is expressed in the inclusion bodies, as is the case in many instances, these can be washed in any of several solutions to remove some of the contaminating host proteins, then solubilized in solutions containing high concentrations of urea (e.g., 8M) or chaotropic agents such as guanidine hydrochloride in the presence of reducing agents, such as β-mercaptoethanol or DTT (dithiothreitol).

[0180] Under some circumstances, it may be advantageous to incubate the proteinaceous molecule for several hours under conditions suitable for the proteinaceous molecule to undergo a refolding process into a conformation which more closely resembles that of the native proteinaceous molecule. Such conditions generally include low proteinaceous molecule concentrations, less than 500 mg/ml, low levels of reducing agent, concentrations of urea less than 2 M and often the presence of reagents such as a mixture of reduced and oxidized glutathione which facilitate the interchange of disulfide bonds within the proteinaceous molecule.

[0181] The refolding process can be monitored, for example, by SDS-PAGE, or with antibodies specific for the native molecule (which can be obtained from animals vaccinated with the native molecule or smaller quantities of recombinant proteinaceous molecule). Following refolding, the proteinaceous molecule can then be purified further and separated from the refolding mixture by chromatography on any of several supports including ion exchange resins, gel permeation resins or on a variety of affinity columns.

[0182] For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used. This plasmid already contains the trp1 gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1. The presence of the trp1 lesion as a characteristic of the yeast host cell genome then provides an effective enviromuent for detecting transformation by growth in the absence of tryptophan.

[0183] Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3′ of the sequence desired to be expressed to provide polyadenylation of the MRNA and termination.

[0184] Other suitable promoters, which have the additional advantage of transcription controlled by growth conditions, include the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.

[0185] In addition to micro-organisms, cultures of cells derived from multicellular organisms may also be used as hosts. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture. In addition to mammalian cells, these include insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus); and plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing one or more sGC protein, polypeptide or peptide coding sequences.

[0186] In a useful insect system, Autograph californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The sGC protein, polypeptide or peptide coding sequences are cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of the coding sequences results in the inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed (e.g., U.S. Pat. No. 4,215,051, Smith, incorporated herein by reference).

[0187] Examples of useful mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines. In addition, a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of proteinaceous products may be important for the function of the proteinaceous molecule.

[0188] Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteinaceous molecules. Appropriate cells lines or host systems can be chosen to ensure the correct modification and processing of the foreign proteinaceous molecule expressed.

[0189] Expression vectors for use in mammalian cells ordinarily include an origin of replication (as necessary), a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences. The origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.

[0190] The promoters may be derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Further, it is also possible, and may be desirable, to utilize promoter or control sequences normally associated with the sGC gene, provided such control sequences are compatible with the host cell systems.

[0191] A number of viral based expression systems may be utilized, for example, commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40). The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication. Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the HindIII site toward the Bg1 I site located in the viral origin of replication.

[0192] In cases where an adenovirus is used as an expression vector, the coding sequences may be ligated to an adenovirus transcription/ translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1, E3, or E4) will result in a recombinant virus that is viable and capable of expressing sGC proteins, polypeptides or peptides in infected hosts.

[0193] Specific initiation signals may also be required for efficient translation of sGC protein, polypeptide or peptide coding sequences. These signals include the ATG initiation codon and adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may additionally need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be in-frame (or in-phase) with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements and transcription terminators.

[0194] In eukaryotic expression, one will also typically desire to incorporate into the transcriptional unit an appropriate polyadenylation site (e.g., 5′-AATAAA-3′) if one was not contained within the original cloned segment. Typically, the poly A addition site is placed about 30 to 2000 nucleotides “downstream” of the termination site of the proteinaceous molecule at a position prior to transcription termination.

[0195] For long-term, high-yield production of a recombinant sGC protein, polypeptide or peptide, stable expression is preferred. For example, cell lines that stably express constructs encoding an sGC protein, polypeptide or peptide may be engineered. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with vectors controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.

[0196] A number of selection systems may be used, including, but not limited to, the herpes simplex virus thymidine kinase (tk), hypoxanthine-guanine phosphoribosyltransferase (hgprt) and adenine phosphoribosyltransferase (aprt) genes, in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dihydrofolate reductase (dhfr), that confers resistance to methotrexate; gpt, that confers resistance to mycophenolic acid; neomycin (neo), that confers resistance to the aminoglycoside G-418; and hygromycin (hygro), that confers resistance to hygromycin.

[0197] Animal cells can be propagated in vitro in two modes: as non-anchorage dependent cells growing in suspension throughout the bulk of the culture or as anchorage-dependent cells requiring attachment to a solid substrate for their propagation (i.e., a monolayer type of cell growth).

[0198] Non-anchorage dependent or suspension cultures from continuous established cell lines are the most widely used means of large scale production of cells and cell products. However, suspension cultured cells have limitations, such as tumorigenic potential and lower proteinaceous molecule production than adherent cells.

[0199] Large scale suspension culture of mammalian cells in stirred tanks is a common method for production of recombinant proteinaceous molecules. Two suspension culture reactor designs are in wide use—the stirred reactor and the airlift reactor. The stirred design has successfully been used on an 8000 liter capacity for the production of interferon. Cells are grown in a stainless steel tank with a height-to-diameter ratio of 1:1 to 3:1. The culture is usually mixed with one or more agitators, based on bladed disks or marine propeller patterns. Agitator systems offering less shear forces than blades have been described. Agitation may be driven either directly or indirectly by magnetically coupled drives. Indirect drives reduce the risk of microbial contamination through seals on stirrer shafts.

[0200] The airlift reactor, also initially described for microbial fermentation and later adapted for mammalian culture, relies on a gas stream to both mix and oxygenate the culture. The gas stream enters a riser section of the reactor and drives circulation. Gas disengages at the culture surface, causing denser liquid free of gas bubbles to travel downward in the downcomer section of the reactor. The main advantage of this design is the simplicity and lack of need for mechanical mixing. Typically, the height-to-diameter ratio is 10:1. The airlift reactor scales up relatively easily, has good mass transfer of gases and generates relatively low shear forces.

[0201] It is contemplated that the sGC proteins, polypeptides or peptides of the invention may be “overexpressed”, i.e., expressed in increased levels relative to its natural expression in cells. Such overexpression may be assessed by a variety of methods, including radio-labeling and/or proteinaceous molecule purification. However, simple and direct methods are preferred, for example, those involving SDS/PAGE and proteinaceous composition staining or western blotting, followed by quantitative analyses, such as densitometric scanning of the resultant gel or blot. A specific increase in the level of the recombinant protein, polypeptide or peptide in comparison to the level in natural cells is indicative of overexpression, as is a relative abundance of the specific proteinaceous molecule in relation to the other proteins produced by the host cell and, e.g., visible on a gel.

[0202] IV. Methods of Gene Transfer

[0203] In order to mediate the effect of transgene expression in a cell, it will be necessary to transfer the expression constructs (e.g., a therapeutic construct) of the present invention into a cell. Such transfer may employ viral or non-viral methods of gene transfer. This section provides a discussion of methods and compositions of gene or nucleic acid transfer.

[0204] 1. Viral Vector-Mediated Transfer

[0205] The mammalian sGC nucleic acids are incorporated into an adenoviral infectious particle to mediate gene transfer to a cell. Additional expression constructs encoding other therapeutic agents as described herein may also be transferred via viral transduction using infectious viral particles, for example, by transformation with an adenovirus vector of the present invention as described herein below. Alternatively, retroviral or bovine papilloma virus may be employed, both of which permit permanent transformation of a host cell with a gene(s) of interest. Thus, in one example, viral infection of cells is used in order to deliver therapeutically significant genes to a cell. Typically, the virus simply will be exposed to the appropriate host cell under physiologic conditions, permitting uptake of the virus. Though adenovirus is exemplified, the present methods may be advantageously employed with other viral vectors, as discussed below.

[0206] Adenovirus. Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized DNA genome, ease of manipulation, high titer, wide target-cell range, and high infectivity. The roughly 36 kB viral genome is bounded by 100-200 base pair (bp) inverted terminal repeats (ITR), in which are contained cis-acting elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome that contain different transcription units are divided by the onset of viral DNA replication.

[0207] The El region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shut off (Renan, 1990). The products of the late genes (L1, L2, L3, L4 and L5), including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP (located at 16.8 map units) is particularly efficient during the late phase of infection, and all the mRNAs issued from this promoter possess a 5′ tripartite leader (TL) sequence which makes them preferred mRNAs for translation.

[0208] In order for adenovirus to be optimized for gene therapy, it is necessary to maximize the carrying capacity so that large segments of DNA can be included. It also is very desirable to reduce the toxicity and immunologic reaction associated with certain adenoviral products. The two goals are, to an extent, coterminous in that elimination of adenoviral genes serves both ends. By practice of the present invention, it is possible achieve both these goals while retaining the ability to manipulate the therapeutic constructs with relative ease.

[0209] The large displacement of DNA is possible because the cis elements required for viral DNA replication all are localized in the inverted terminal repeats (ITR) (100-200 bp) at either end of the linear viral genome. Plasmids containing ITR's can replicate in the presence of a non-defective adenovirus (Hay et al., 1984). Therefore, inclusion of these elements in an adenoviral vector should permit replication.

[0210] In addition, the packaging signal for viral encapsidation is localized between 194-385 bp (0.5-1.1 map units) at the left end of the viral genome (Hearing et al, 1987). This signal mimics the protein recognition site in bacteriophage λ DNA where a specific sequence close to the left end, but outside the cohesive end sequence, mediates the binding to proteins that are required for insertion of the DNA into the head structure. E1 substitution vectors of Ad have demonstrated that a 450 bp (0-1.25 map units) fragment at the left end of the viral genome could direct packaging in 293 cells (Levrero et al., 1991).

[0211] Previously, it has been shown that certain regions of the adenoviral genome can be incorporated into the genome of mammalian cells and the genes encoded thereby expressed. These cell lines are capable of supporting the replication of an adenoviral vector that is deficient in the adenoviral function encoded by the cell line. There also have been reports of complementation of replication deficient adenoviral vectors by “helping” vectors, e.g., wild-type virus or conditionally defective mutants.

[0212] Replication-deficient adenoviral vectors can be complemented, in trans, by helper virus. This observation alone does not permit isolation of the replication-deficient vectors, however, since the presence of helper virus, needed to provide replicative functions, would contaminate any preparation. Thus, an additional element was needed that would add specificity to the replication and/or packaging of the replication-deficient vector. That element, as provided for in the present invention, derives from the packaging function of adenovirus.

[0213] It has been shown that a packaging signal for adenovirus exists in the left end of the conventional adenovirus map (Tibbetts, 1977). Later studies showed that a mutant with a deletion in the E1A (194-358 bp) region of the genome grew poorly even in a cell line that complemented the early (E1A) function (Hearing and Shenk, 1983). When a compensating adenoviral DNA (0-353 bp) was recombined into the right end of the mutant, the virus was packaged normally. Further mutational analysis identified a short, repeated, position-dependent element in the left end of the Ad5 genome. One copy of the repeat was found to be sufficient for efficient packaging if present at either end of the genome, but not when moved towards the interior of the Ad5 DNA molecule (Hearing et al., 1987).

[0214] By using mutated versions of the packaging signal, it is possible to create helper viruses that are packaged with varying efficiencies. Typically, the mutations are point mutations or deletions. When helper viruses with low efficiency packaging are grown in helper cells, the virus is packaged, albeit at reduced rates compared to wild-type virus, thereby permitting propagation of the helper. When these helper viruses are grown in cells along with virus that contains wild-type packaging signals, however, the wild-type packaging signals are recognized preferentially over the mutated versions. Given a limiting amount of packaging factor, the virus containing the wild-type signals are packaged selectively when compared to the helpers. If the preference is great enough, stocks approaching homogeneity should be achieved.

[0215] Retrovirus. The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins.

[0216] The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes—gag, pol and env—that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene, termed ψ, functions as a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5′ and 3′ ends of the viral genome. These contain strong promoter and enhancer sequences and also are required for integration in the host cell genome (Coffin, 1990).

[0217] In order to construct a retroviral vector, a nucleic acid encoding a promoter is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol and env genes but without the LTR and ψ components is constructed (Mann et al., 1983). When a recombinant plasmid containing a human cDNA, together with the retroviral LTR and ψ sequences is introduced into this cell line (by calcium phosphate precipitation for example), the ψ sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containing the recombinant retroviruses is collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types.

[0218] However, integration and stable expression of many types of retroviruses require the division of host cells (Paskind et al., 1975).

[0219] An approach designed to allow specific targeting of retrovirus vectors recently was developed based on the chemical modification of a retrovirus by the chemical addition of galactose residues to the viral envelope. This modification could permit the specific infection of cells such as hepatocytes via asialoglycoprotein receptors, should this be desired.

[0220] A different approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin (Roux et al., 1989). Using antibodies against major histocompatibility complex class I and class II antigens, the infection of a variety of human cells that bore those surface antigens was demonstrated with an ecotropic virus in vitro (Roux et al., 1989).

[0221] Adeno-associated Virus. AAV utilizes a linear, single-stranded DNA of about 4700 base pairs. Inverted terminal repeats flank the genome. Two genes are present within the genome, giving rise to a number of distinct gene products. The first, the cap gene, produces three different virion proteins (VP), designated VP-1, VP-2 and VP-3. The second, the rep gene, encodes four non-structural proteins (NS). One or more of these rep gene products is responsible for transactivating AAV transcription.

[0222] The three promoters in AAV are designated by their location, in map units, in the genome. These are, from left to right, p5, p19 and p40. Transcription gives rise to six transcripts, two initiated at each of three promoters, with one of each pair being spliced. The splice site, derived from map units 42-46, is the same for each transcript. The four non-structural proteins apparently are derived from the longer of the transcripts, and three virion proteins all arise from the smallest transcript.

[0223] AAV is not associated with any pathologic state in humans. Interestingly, for efficient replication, AAV requires “helping” functions from viruses such as herpes simplex virus I and II, cytomegalovirus, pseudorabies virus and, of course, adenovirus. The best characterized of the helpers is adenovirus, and many “early” functions for this virus have been shown to assist with AAV replication. Low level expression of AAV rep proteins is believed to hold AAV structural expression in check, and helper virus infection is thought to remove this block.

[0224] The terminal repeats of the AAV vector can be obtained by restriction endonuclease digestion of AAV or a plasmid such as p201, which contains a modified AAV genome (Samulski et al., 1987), or by other methods known to the skilled artisan, including but not limited to chemical or enzymatic synthesis of the terminal repeats based upon the published sequence of AAV. The ordinarily skilled artisan can determine, by well-known methods such as deletion analysis, the minimum sequence or part of the AAV ITRs which is required to allow function, i.e., stable and site-specific integration. The ordinarily skilled artisan also can determine which minor modifications of the sequence can be tolerated while maintaining the ability of the terminal repeats to direct stable, site-specific integration.

[0225] AAV-based vectors have proven to be safe and effective vehicles for gene delivery in vitro, and these vectors are being developed and tested in pre-clinical and clinical stages for a wide range of applications in potential gene therapy, both ex vivo and in vivo (Carter and Flotte, 1996; Chatteijee et al., 1995; Ferrari et al., 1996; Fisher et al., 1996; Flotte et al., 1993; Goodman et al., 1994; Kaplitt et al., 1994; 1996, Kessler et al., 1996; Koeberl et al., 1997; Mizukami et al., 1996; Xiao et al., 1996).

[0226] AAV-mediated efficient gene transfer and expression in the lung has led to clinical trials for the treatment of cystic fibrosis (Carter and Flotte, 1996; Flotte et al., 1993). Similarly, the prospects for treatment of muscular dystrophy by AAV-mediated gene delivery of the dystrophin gene to skeletal muscle, of Parkinson's disease by tyrosine hydroxylase gene delivery to the brain, of hemophilia B by Factor IX gene delivery to the liver, and potentially of myocardial infarction by vascular endothelial growth factor gene to the heart, appear promising since AAV-mediated transgene expression in these organs has recently been shown to be highly efficient (Fisher et al., 1996; Flotte et al., 1993; Kaplitt et al., 1994; 1996; Koeberl et al, 1997; McCown et al., 1996; Ping et al., 1996; Xiao et al., 1996).

[0227] Other Viral Vectors. Other viral vectors may be employed as expression constructs in the present invention. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988) canary pox virus, and herpes viruses may be employed. These viruses offer several features for use in gene transfer into various mammalian cells.

[0228] 2. Non-viral Transfer

[0229] DNA constructs of the present invention are generally delivered to a cell, in certain situations, the nucleic acid to be transferred is non-infectious, and can be transferred using non-viral methods.

[0230] Several non-viral methods for the transfer of expression constructs into cultured mammalian cells are contemplated by the present invention. Suitable methods for nucleic acid delivery for transformation of an organelle, a cell, a tissue or an organism for use with the current invention are believed to include virtually any method by which a nucleic acid (e.g., DNA) can be introduced into an organelle, a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harlan and Weintraub, 1985; U.S. Pat. No. 5,789,215, incorporated herein by reference); by electroporation (U.S. Pat. No. 5,384,253, incorporated herein by reference; Tur-Kaspa et al., 1986; Potter et al., 1984); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991) and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988); by microprojectile bombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and 5,464,765, each incorporated herein by reference); by Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and 5,563,055, each incorporated herein by reference); or by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos. 4,684,611 and 4,952,500, each incorporated herein by reference); by desiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985). Through the application of techniques such as these, organelle(s), cell(s), tissue(s) or organism(s) may be stably or transiently transformed.

[0231] Once the construct has been delivered into the cell the nucleic acid encoding the therapeutic gene may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the therapeutic gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.

[0232] In a particular embodiment of the invention, the expression construct may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). The addition of DNA to cationic liposomes causes a topological transition from liposomes to optically birefringent liquid-crystalline condensed globules (Radler et al., 1997). These DNA-lipid complexes are potential non-viral vectors for use in gene therapy.

[0233] Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful. Using the P-lactamase gene, Wong et al. (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa, and hepatoma cells. Nicolau et al. (1987) accomplished successful liposome-mediated gene transfer in rats after intravenous injection. Also included are various commercial approaches involving “lipofection” technology.

[0234] In certain embodiments of the invention, the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell zmembrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments, the liposome may be complexed or employed in conjunction with nuclear nonhistone chromosomal proteins (HMG-1) (Kato et al., 1991). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In that such expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention.

[0235] Other vector delivery systems which can be employed to deliver a nucleic acid encoding a therapeutic gene into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993).

[0236] Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferring (Wagner et al., 1990). Recently, a synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle (Ferkol et al., 1993; Perales et al., 1994) and epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells (Myers, EPO 0273085).

[0237] In other embodiments, the delivery vehicle may comprise a ligand and a liposome. For example, Nicolau et al. (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes. Thus, it is feasible that a nucleic acid encoding a therapeutic gene also may be specifically delivered into a cell type such as prostate, epithelial or tumor cells, by any number of receptor-ligand systems with or without liposomes. For example, the human prostate-specific antigen (Watt et al., 1986) may be used as the receptor for mediated delivery of a nucleic acid in prostate tissue.

[0238] In another embodiment of the invention, the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is applicable particularly for transfer in vitro, however, it may be applied for in vivo use as well. Dubensky et al (1984) successfully injected polyomavirus DNA in the form of CaPO₄ precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of CaPO₄ precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding a CAM may also be transferred in a similar manner in vivo and express CAM.

[0239] Another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al., 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al., 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.

[0240] 3. Methods of Making Transgenic Animals

[0241] As noted above, a particular embodiment of the present invention provides transgenic animals that contain an inactive SGC.

[0242] Although the present discussion refers to transgenic mice, it is understood that mice are merely exemplary model animal, and any other mammalian animal routinely used as model animal (e.g., rat, guinea pig, rabbit, cats, dogs, pigs and the like) may be generated using the technology described herein. In a general aspect, a transgenic animal is produced by the integration of a given transgene into the genome in a manner that permits the expression of the transgene. The terms “animal” and “non-human animal”, as used herein, include all vertebrate animals, except humans. It also includes individual animals in all stages of development, including embryonic and fetal stages. A “transgenic animal” is any animal containing one or more cells bearing genetic information received, directly or indirectly, by deliberate genetic manipulation at the subcellular level. The genetic manipulation can be performed by any method of introducing genetic material to a cell, including, but not limited to, microinjection, infection with a recombinant virus, particle bombardment or electroporation. The term is not intended to encompass classical cross-breeding or in vitro fertilization, but rather is meant to encompass animals in which one or more cells receive a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA. The genetic information may be foreign to the species of animal to which the recipient belongs, foreign only to the individual recipient, or genetic information already possessed by the recipient expressed at a different level, a different time, or in a different location than the native gene.

[0243] Methods for producing transgenic animals are generally described by Wagner and Hoppe (U.S. Pat. No. 4,873,191; which is incorporated herein by reference), Brinster et al. 1985; which is incorporated herein by reference in its entirety) and in “Manipulating the Mouse Embryo; A Laboratory Manual” 2nd edition (eds., Hogan, Beddington, Costantimi and Long, Cold Spring Harbor Laboratory Press, 1994; which is incorporated herein by reference in its entirety).

[0244] Typically, a gene flanked by genomic sequences is transferred by microinjection into a fertilized egg. The microinjected eggs are implanted into a host female, and the progeny are screened for the expression of the transgene. Transgenic animals may be produced from the fertilized eggs from a number of animals including, but not limited to reptiles, amphibians, birds, mammals, and fish. Within a particularly preferred embodiment, transgenic mice are generated which express a mutant form of the SGC polypeptide which lacks the carboxy-terminal domain of wild-type SGC.

[0245] DNA clones for microinjection can be prepared by any means known in the art. For example, DNA clones for microinjection can be cleaved with enzymes appropriate for removing the bacterial plasmid sequences, and the DNA fragments electrophoresed on 1% agarose gels in TBE buffer, using standard techniques. The DNA bands are visualized by staining with ethidium bromide, and the band containing the expression sequences is excised. The excised band is then placed in dialysis bags containing 0.3 M sodium acetate, pH 7.0. DNA is electroeluted into the dialysis bags, extracted with a 1:1 phenol:chloroform solution and precipitated by two volumes of ethanol. The DNA is redissolved in 1 ml of low salt buffer (0.2 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) and purified on an Elutip-D™ column. The column is first primed with 3 ml of high salt buffer (1 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) followed by washing with 5 ml of low salt buffer. The DNA solutions are passed through the column three times to bind DNA to the column matrix. After one wash with 3 ml of low salt buffer, the DNA is eluted with 0.4 ml high salt buffer and precipitated by two volumes of ethanol. DNA concentrations are measured by absorption at 260 nm in a UV spectrophotometer. For microinjection, DNA concentrations are adjusted to 3 μg/ml in 5 mM Tris, pH 7.4 and 0.1 mM EDTA.

[0246] Other methods for purification of DNA for microinjection are described in Hogan et al. Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1986), in Palmiter et al. Nature 300:611 (1982); in The Qiagenologist, Application Protocols, 3rd edition, published by Qiagen, Inc., Chatsworth, Calif.; and in Sambrook et al. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).

[0247] Female mice are induced to superovulate, e.g., by using an injection of pregnant mare serum gonadotropin (PMSG; Sigma) followed, 48 hours later, by an injection of human chronic gonadotropin (hCG; Sigma). Females are placed with males immediately after hCG injection. Twenty-one hours after hCG injection, the mated females are sacrificed by CO₂ asphyxiation or cervical dislocation and embryos are recovered from excised oviducts and placed in Dulbecco's phosphate buffered saline with 0.5% bovine serum albumin (BSA; Sigma). Surrounding cumulus cells are removed with hyaluronidase (1 mg/ml). Pronuclear embryos are then washed and placed in Earle's balanced salt solution containing 0.5% BSA (EBSS) in a 37.5° C. incubator with a humidified atmosphere at 5% CO₂, 95% air until the time of injection. Embryos can be implanted at the two-cell stage.

[0248] 25 μg of a Sa1I-linearized SGC targeting vector is electroporated into 1×10⁷ embryonic stem (ES) cells. After a suitable period of incubation, e.g., 36 hr, the transfected cells are then selected using G418 and FIAU. The G418-FIAU-resistant ES colonies are picked into 96-well plates (Ramirez-Solis et al., 1993). Positive ES clones are injected into C57BL/6 blastocysts and transferred into pseudopregnant ICR female recipients. At the time of embryo transfer, the recipient females are anesthetized with an intraperitoneal injection of 0.015 ml of 2.5% avertin per gram of body weight. The oviducts are exposed by a single midline dorsal incision. An incision is then made through the body wall directly over the oviduct. The ovarian bursa is then torn with watchmakers forceps. Embryos to be transferred are placed in DPBS (Dulbecco's phosphate buffered saline) and in the tip of a transfer pipet (about 10 to 12 embryos). The pipet tip is inserted into the infundibulum and the embryos transferred. After the transfer, the incision is closed by two sutures.

[0249] The resulting male chimeras are bred with C57BL/6 females. Germline transmission can be screened by using a phenotype, such as coat color and confirmed by Southern analysis. To obtain the targeted SGC allele in an inbred 129/Sv background, a male chimera is directly bred with 129/Sv female mice.

[0250] As noted above, transgenic animals and cell lines derived from such animals may find use in certain testing experiments. In this regard, transgenic animals and cell lines capable of expressing a mutant SGC may be exposed to test substances. These test substances can be screened for the ability to restore TGFβ signaling, and alter the growth of the cell lines and/or the colorectal, neurofibrosarcoma, glioma, astrocytoma, lung cancer or pancreatic tumors in the transgenic animals. Compounds identified by such procedures will be useful in the treatment of colorectal or other cancers involving an aberrant TGFβ-signal caused by altered or dysfunctional SGC expression and/or activity. Thus the compounds identified may be used to prevent, treat, ameliorate tumor growth, cell proliferation, decrease tumor size, or otherwise have a beneficial effect against colorectal cancer or other cancers modeled by the animal or cell lines.

a. ES Cells

[0251] ES cells are obtained from pre-implantation embryos cultured in vitro (Evans et al. 1981; Bradley et al 1984; Gossler et al. 1986; Robertson et al. (1986). Transgenes are introduced into ES cells using a number of means well known to those of skill in the art. The transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal (for a review see Jaenisch, 1988).

[0252] Once the DNA is introduced, e.g., by electroporation (Troneguzzo et al., 1988; Quillet et al., 1988; Machy et al., 1988), the cells are cultured under conventional conditions well known in the art. In order to facilitate the recovery of those cells which have received the DNA molecule containing the desired gene sequence, it is preferable to introduce the DNA containing the desired gene sequence in combination with a second gene sequence which would contain a detectable marker gene sequence. For the purposes of the present invention, any gene sequence whose presence in a cell permits one to recognize and clonally isolate the cell may be employed as a detectable (selectable) marker gene sequence. The presence of the detectable (selectable) marker sequence in a recipient cell may be recognized by PCR, by detection of radiolabeled nucleotides, or by other assays of detection which do not require the expression of the detectable marker sequence. Typically, the detectable marker gene sequence will be expressed in the recipient cell, and will result in a selectable phenotype. Selectable markers are well known to those of skill in the art. Some examples include the hprt gene (Littlefield, 1964), the neo gene, the tk (thyroidinc kinase) gene of herpes simplex virus (Giphart-Gassler et al., 1989), or other genes which confer resistance to amino acid or nucleoside analogues, or antibiotics, etc.

[0253] Any ES cell may be used in accordance with the present invention. It is, however, preferred to use primary isolates of ES cells. Such isolates may be obtained directly from embryos such as the CCE cell line disclosed by Robertson (1989), or from the clonal isolation of ES cells from the CCE cell line (Schwartzberg et al., 1989). Such clonal isolation may be accomplished according to the method of Robertson (1987). The purpose of such clonal propagation is to obtain ES cells which have a greater efficiency for differentiating into an animal. Clonally selected ES cells are approximately 10-fold more effective in producing transgenic animals than the progenitor cell line CCE.

b. Homologous recombination

[0254] Homologous recombination (Koller and Smithies, 1992), directs the insertion of the transgene to a specific location. This technique allows the precise modification of existing genes, and overcomes the problems of positional effects and insertional inactivation observed with transgenic animals generated by pronuclear injection or use of viral vectors. Additionally, it allows the inactivation of specific genes as well as the replacement of one gene for another. In particular embodiments, the DNA segment comprises two selected DNA regions that flank the SGC coding region, thereby directing the homologous recombination of the coding region into the genomic DNA of a non-human animal species.

[0255] Thus, a preferred method for the delivery of transgenic constructs involves the use of homologous recombination, or “knock-out technology”. Homologous recombination relies, like antisense, on the tendency of nucleic acids to base pair with complementary sequences. In this instance, the base pairing serves to facilitate the interaction of two separate nucleic acid molecules so that strand breakage and repair can take place. In other words, the “homologous” aspect of the method relies on sequence homology to bring two complementary sequences into close proximity, while the “recombination” aspect provides for one complementary sequence to replace the other by virtue of the breaking of certain bonds and the formation of others.

[0256] Put into practice, homologous recombination is used as follows. First, the target gene is selected within the host cell. Sequences homologous to the target gene are then included in a genetic construct, along with some mutation that will render the target gene inactive (stop codon, interruption, and the like). The homologous sequences flanking the inactivating mutation are said to “flank” the mutation. Flanking, in this context, simply means that target homologous sequences are located both upstream (5′) and downstream (3′) of the mutation. These sequences should correspond to some sequences upstream and downstream of the target gene. The construct is then introduced into the cell, thus permitting recombination between the cellular sequences and the construct.

[0257] As a practical matter, the genetic construct will normally act as far more than a vehicle to interrupt the gene. For example, it is important to be able to select for recombinants and, therefore, it is common to include within the construct a selectable marker gene. This gene permits selection of cells that have integrated the construct into their genomic DNA by conferring resistance to various biostatic and biocidal drugs. In addition, a heterologous gene that is to be expressed in the cell also may advantageously be included within the construct. The arrangement might be as follows:

vector·5′-flanking sequence·heterologous gene·selectable marker gene·flanking sequence-3·-vector

[0258] Thus, using this kind of construct, it is possible, in a single recombinatorial event, to (i) “knock out” an endogenous gene, (ii) provide a selectable marker for identifying such an event and (iii) introduce a transgene for expression.

[0259] Another refinement of the homologous recombination approach involves the use of a “negative” selectable marker. This marker, unlike the selectable marker, causes death of cells which express the marker. Thus, it is used to identify undesirable recombination events. When seeking to select homologous recombinants using a selectable marker, it is difficult in the initial screening step to identify proper homologous recombinants from recombinants generated from random, non-sequence specific events. These recombinants also may contain the selectable marker gene and may express the heterologous protein of interest, but will, in all likelihood, not have the desired “knock out” phenotype. By attaching a negative selectable marker to the construct, but outside of the flanking regions, one can select against many random recombination events that will incorporate the negative selectable marker. Homologous recombination should not introduce the negative selectable marker, as it is outside of the flanking sequences. Examples of processes that use negative selection to enrich for homologous recombination include the disruption of targeted genes in embryonic stem cells or transformed cell lines (Mortensen, 1993; Willnow and Herz, 1994) and the production of recombinant virus such as adenovirus (Imler et al., 1995).

[0260] Since the frequency of gene targeting is heavily influenced by the origin of the DNA being used for targeting, it is beneficial to obtain DNA that is as similar (isogenic) to the cells being targeted as possible. One way to accomplish this is by isolation of the region of interest from genomic DNA from a single colony by long range PCR. Using long range PCR it is possible to isolate fragments of 7-12 kb from small amounts of starting DNA.

[0261] Gene trapping is a useful technique suitable for use with the present invention. This refers to the utilization of the endogenous regulatory regions present in the chromosomal DNA to activate the incoming transgene. In this way expression of the transgene is absent or minimized when the transgene inserts in a random location. However, when homologous recombination occurs the endogenous regulatory region are placed in apposition to the incoming transgene, which results in expression of the transgene.

C. Site Specific Recombination

[0262] Members of the integrase family are proteins that bind to a DNA recognition sequence, and are involved in DNA recognition, synapsis, cleavage, strand exchange, and religation. Currently, the family of integrases includes 28 proteins from bacteria, phage, and yeast which have a common invariant His-Arg-Tyr triad (Abremski and Hoess, 1992). Four of the most widely used site-specific recombination systems for eukaryotic applications include: Cre-loxP from bacteriophage P1 (Austin et al., 1981); FLP-FRT from the 2μ plasmid of Saccharomyces cerevisiae (Andrews et al., 1985); R-RS from Zygosaccharomyces rouxii (Maeser and Kahmann, 1991) and gin-gix from bacteriophage Mu (Onouchi et al., 1995). The Cre-loxP and FLP-FRT systems have been developed to a greater extent than the latter two systems. The R-RS system, like the Cre-loxP and FLP-FRT systems, requires only the protein and its recognition site. The Gin recombinase selectively mediates DNA inversion between two inversely oriented recombination sites (gix) and requires the assistance of three additional factors: negative supercoiling, an enhancer sequence and its binding protein Fis.

[0263] The present invention contemplates the use of the Cre/Lox site-specific recombination system (Sauer, 1993, available through Gibco/BRL, Inc., Gaithersburg, Md.) to rescue specific genes out of a genome, and to excise specific transgenic constructs from the genome. The Cre (causes recombination)-lox P (locus of crossing-over(x)) recombination system, isolated from bacteriophage P1, requires only the Cre enzyme and its loxP recognition site on both partner molecules (Stemberg and Hamilton, 1981). The loxp site consists of two symmetrical 13 bp protein binding regions separated by an 8 bp spacer region, which is recognized by the Cre recombinase, a 35 kDa protein. Nucleic acid sequences for loxP (Hoess et al., 1982) and Cre (Stemberg et al., 1986) are known. If the two lox P sites are cis to each other, an excision reaction occurs; however, if the two sites are trans to one another, an integration event occurs. The Cre protein catalyzes a site-specific recombination event. This event is bidirectional, i.e., Cre will catalyze the insertion of sequences at a LoxP site or excise sequences that lie between two LoxP sites. Thus, if a construct for insertion also has flanking LoxP sites, introduction of the Cre protein, or a polynucleotide encoding the Cre protein, into the cell will catalyze the removal of the construct DNA. This technology is enabled in U.S. Pat. No. 4,959,317, which is hereby incorporated by reference in its entirety.

[0264] An initial in vivo study in bacteria showed that the Cre excises loxP-flanked DNA extrachromosomally in cells expressing the recombinase (Abremski et al., 1983). A major question regarding this system was whether site-specific recombination in eukaryotes could be promoted by a bacterial protein. However, Sauer (1987) showed that the system excises DNA in S. cerevisiae with the same level of efficiency as in bacteria.

[0265] Further studies with the Cre-loxP system, in particular the ES cells system in mice, has demonstrated the usefulness of the excision reaction for the generation of unique transgenic animals. Homologous recombination followed by Cre-mediated deletion of a loxP-flanked neo-tk cassette was used to introduce mutations into ES cells. This strategy was repeated for a total of 4 rounds in the same line to alter both alleles of the rep-3 and mMsh2 loci, genes involved in DNA mismatch repair (Abuin and Bradley, 1996). Similarly, a transgene which consists of the 35S promoter/luciferase gene/loxP/35S promoter/hpt gene/loxP (luc⁺hyg⁺) was introduced into tobacco. Subsequent treatment with Cre causes the deletion of the hyg gene (luc⁺hyg^(S)) at 50% efficiency (Dale and Ow, 1991). Transgenic mice which have the Ig light chain κ constant region targeted with a loxP-flanked neo gene were bred to Cre-producing mice to remove the selectable marker from the early embryo (Lakso et al., 1996). This general approach for removal of markers stems from issues raised by regulatory groups and consumers concerned about the introduction of new genes into a population.

[0266] An analogous system contemplated for use in the present invention is the FLP/FRT system. This system was used to target the histone 4 gene in mouse ES cells with a FRT-flanked neo cassette followed by deletion of the marker by FLP-mediated recombination. The FLP protein could be obtained from an inducible promoter driving the FLP or by using the protein itself (Wigley et al., 1994).

[0267] The present invention also contemplates the use of recombination activating genes (RAG) 1 and 2 to excise specific transgenic constructs from the genome, as well as to rescue specific genes from the genome. RAG-1 (GenBank accession number M29475) and RAG-2 (GenBank accession numbers M64796 and M33828) recognize specific recombination signal sequences (RSSs) and catalyze V(D)J recombination required for the assembly of immunoglobulin and T cell receptor genes (Schatz et al., 1989; Oettinger et al., 1990; Cumo and Oettinger, 1994). Transgenic expression of RAG-1 and RAG-2 proteins in non-lymphoid cells supports V(D)J recombination of reporter substrates (Oettinger et al., 1990). For use in the present invention, the transforming construct of interest is engineered to contain flanking RSSs. Following transformation, the transforming construct that is internal to the RSSs can be deleted from the genome by the transient expression of RAG-1 and RAG-2 in the transformed cell.

[0268] V. sGC Proteins, Polypeptides, and Peptides

[0269] The present invention also provides purified, and in preferred embodiments, substantially purified mammalian sGC proteins, polypeptides, or peptides. The term “purified mammalian sGC proteins, polypeptides, or peptides” as used herein, is intended to refer to an sGC proteinaceous composition, isolatable from mammalian cells or recombinant host cells, wherein the sGC protein, polypeptide, or peptide is purified to any degree relative to its naturally-obtainable state, i.e., relative to its purity within a cellular extract. A purified sGC protein, polypeptide, or peptide therefore also refers to a wild-type or mutant sGC protein, polypeptide, or peptide free from the environment in which it naturally occurs.

[0270] The sGC proteins may be full length proteins, such as being 826 amino acids in length. The sGC proteins, polypeptides and peptides may also be less then full length proteins, such as individual polypeptide, domains, regions or even epitopic peptides. Where less than full length sGC proteins are concerned the most preferred will be those containing predicted immunogenic sites and those containing the functional domains identified herein.

[0271] Encompassed by the invention are proteinaceous segments of relatively small peptides, such as, for example, peptides of from about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 35, about 40, about 45, to about 50 amino acids in length, and more preferably, of from about 15 to about 30 amino acids in length; as set forth in SEQ ID NO: 2, and also larger polypeptides of from about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 220, about 240, about 260, about 280, about 300, about 320, about 340, about 360, about 380, about 400, about 420, about 440, about 460, about 480, about 500, about 520, about 540, about 560, about 580, about 600, about 620, about 640, about 660, about 680, about 700, about 720, about 740, about 760, about 780, about 800, about 820, up to and including proteins corresponding to the full-length sequences set forth in SEQ ID NO: 2.

[0272] Generally, “purified” will refer to an sGC protein, polypeptide, or peptide composition that has been subjected to fractionation to remove various non-sGC protein, polypeptide, or peptide, and which composition substantially retains its sGC activity, as may be assessed, assays described herein or would be known to one of skill in the art.

[0273] Where the term “substantially purified” is used, this will refer to a composition in which the sGC protein, polypeptide, or peptide forms the major component of the composition, such as constituting about 50% of the proteinaceous molecules in the composition or more. In preferred embodiments, a substantially purified proteinaceous molecule will constitute more than 60%, 70%, 80%, 90%, 95%, 99% or even more of the proteinaceous molecules in the composition.

[0274] A peptide, polypeptide or protein that is “purified to homogeneity,” as applied to the present invention, means that the peptide, polypeptide or protein has a level of purity where the peptide, polypeptide or protein is substantially free from other proteins and biological components. For example, a purified peptide, polypeptide or protein will often be sufficiently free of other protein components so that degradative sequencing may be performed successfully.

[0275] Various methods for quantifying the degree of purification of sGC proteins, polypeptides, or peptides will be known to those of skill in the art in light of the present disclosure. These include, for example, determnining the specific sGC proteinaceous molecule's activity of a fraction, or assessing the number of proteins, polypeptides and peptides within a fraction by gel electrophoresis. Assessing the number of proteinaceous molecules within a fraction by SDS/PAGE analysis will often be preferred in the context of the present invention as this is straightforward.

[0276] To purify an sGC protein, polypeptide, or peptide a natural or recombinant composition comprising at least some sGC proteins, polypeptides, or peptides will be subjected to fractionation to remove various non-sGC components from the composition. In addition to those techniques described in detail herein below, various other techniques suitable for use in proteinaceous molecule purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite, lectin affinity and other affinity chromatography steps; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques.

[0277] Another example is the purification of an sGC fusion protein using a specific binding partner. Such purification methods are routine in the art. As the present invention provides DNA sequences for sGC proteins, any fusion protein purification method can now be practiced. This is exemplified by the generation of an sGC-glutathione S-transferase fusion protein, expression in E. coli, and isolation to homogeneity using affinity chromatography on glutathione-agarose or the generation of a polyhistidine tag on the N- or C-terminus of the protein, and subsequent purification using Ni-affinity chromatography.

[0278] The exemplary purification methods disclosed herein represent exemplary methods to prepare a substantially purified sGC protein, polypeptide, or polypeptide. These methods are preferred as they result in the substantial purification of the sGC protein, polypeptide or peptide in yields sufficient for further characterization and use. However, given the DNA and proteinaceous molecules provided by the present invention, any purification method can now be employed.

[0279] Although preferred for use in certain embodiments, there is no general requirement that the sGC protein, polypeptide, or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified sGC protein, polypeptide or peptide, which are nonetheless enriched in sGC proteinaceous compositions, relative to the natural state, will have utility in certain embodiments. These include, for example, antibody generation where subsequent screening assays using purified sGC proteinaceous molecules are conducted.

[0280] Methods exhibiting a lower degree of relative purification may have advantages in total recovery of proteinaceous molecule product, or in maintaining the activity of an expressed proteinaceous molecule. Inactive products also have utility in certain embodiments, such as, e.g., in antibody generation.

[0281] VI. Antibodies to sGC Proteins

[0282] A. Epitopic Core Sequences

[0283] Peptides corresponding to one or more antigenic determinants, or “epitopic core regions”, of the sGC proteins of the present invention can also be prepared. Such peptides should generally be at least five or six amino acid residues in length, will preferably be about 10, 15, 20, 25 or about 30 amino acid residues in length, and may contain up to about 35 to about 50 residues or so.

[0284] Synthetic peptides will generally be about 35 residues long, which is the approximate upper length limit of automated peptide synthesis machines, such as those available from Applied Biosystems (Foster City, Calif.). Longer peptides may also be prepared, e.g., by recombinant means.

[0285] U.S. Pat. No. 4,554,101, (Hopp) incorporated herein by reference, teaches the identification and preparation of epitopes from primary amino acid sequences on the basis of hydrophilicity. Through the methods disclosed in Hopp, one of skill in the art would be able to identify epitopes from within an amino acid sequence such as the sGC sequence disclosed herein in SEQ ID NO: 2.

[0286] Numerous scientific publications have also been devoted to the prediction of secondary structure, and to the identification of epitopes, from analyses of amino acid sequences (Chou & Fasman, 1974a,b; 1978a,b, 1979). Any of these may be used, if desired, to supplement the teachings of Hopp in U.S. Pat. No. 4,554,101.

[0287] Moreover, computer programs are currently available to assist with predicting antigenic portions and epitopic core regions of proteinaceous molecules. Examples include those programs based upon the Jameson-Wolf analysis (Jameson & Wolf, 1988; Wolf et al., 1988), the program PepPlot® (Brutlag et al., 1990; Weinberger et al., 1985), and other new programs for proteinaceous molecule tertiary structure prediction (Fetrow and Bryant, 1993). Another commercially available software program capable of carrying out such analyses is MacVector (IBI, New Haven, Conn.).

[0288] In further embodiments, major antigenic determinants of a polypeptide may be identified by an empirical approach in which portions of the gene encoding the polypeptide are expressed in a recombinant host, and the resulting proteinaceous molecules tested for their ability to elicit an immune response. For example, PCR™ can be used to prepare a range of peptides lacking successively longer fragments of the C-terminus of the proteinaceous molecule. The immunoactivity of each of these peptides is determined to identify those fragments or domains of the polypeptide that are immunodominant. Further studies in which only a small number of amino acids are removed at each iteration then allows the location of the antigenic determinants of the polypeptide to be more precisely determined.

[0289] Another method for determining the major antigenic determinants of a polypeptide is the SPOTs™ system (Genosys Biotechnologies, Inc., The Woodlands, Tex.). In this method, overlapping peptides are synthesized on a cellulose membrane, which following synthesis and deprotection, is screened using a polyclonal or monoclonal antibody. The antigenic determinants of the peptides which are initially identified can be further localized by performing subsequent syntheses of smaller peptides with larger overlaps, and by eventually replacing individual amino acids at each position along the immunoreactive peptide.

[0290] Once one or more such analyses are completed, polypeptides are prepared that contain at least the essential features of one or more antigenic determinants. The peptides are then employed in the generation of antisera against the polypeptide. Minigenes or gene fusions encoding these determinants can also be constructed and inserted into expression vectors by standard methods, for example, using PCR™ cloning methodology.

[0291] The use of such small peptides for antibody generation or vaccination typically requires conjugation of the peptide to an immunogenic carrier protein, such as hepatitis B surface antigen, keyhole limpet hemocyanin or bovine serum albumin. Methods for performing this conjugation are well known in the art.

[0292] B. Antibody Generation

[0293] In certain embodiments, the present invention provides antibodies that bind with high specificity to the sGC proteinaceous molecules provided herein. Thus, antibodies that bind to the proteinaceous products of the isolated nucleic acid sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6 are provided. As detailed above, in addition to antibodies generated against the full length proteins, antibodies may also be generated in response to smaller constructs comprising epitopic core regions, including wild-type and mutant epitopes.

[0294] As used herein, the term “antibody” is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally, IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting.

[0295] Monoclonal antibodies (MAbs) are recognized to have certain advantages, e.g., reproducibility and large-scale production, and their use is generally preferred. The invention thus provides monoclonal antibodies of the human, murine, monkey, rat, hamster, rabbit and even chicken origin. Due to the ease of preparation and ready availability of reagents, murine monoclonal antibodies will often be preferred.

[0296] However, “humanized” antibodies are also contemplated, as are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, recombinant and engineered antibodies and fragments thereof. Methods for the development of antibodies that are “custom-tailored” to the patient's dental disease are likewise known and such custom-tailored antibodies are also contemplated.

[0297] The term “antibody” is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab′, Fab, F(ab′)₂, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Means for preparing and characterizing antibodies are also well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference).

[0298] The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic sGC proteinaceous composition in accordance with the present invention and collecting antisera from that immunized animal.

[0299] A wide range of animal species can be used for the production of antisera. Typically the animal used for production of antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.

[0300] As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.

[0301] As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Suitable adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins or synthetic compositions.

[0302] Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12, γ-interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion is also contemplated. MHC antigens may even be used. Exemplary, often preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.

[0303] In addition to adjuvants, it may be desirable to coadminister biologic response modifiers (BRM), which have been shown to upregulate T cell immunity or downregulate suppressor cell activity. Such BRMs include, but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, Pa.); low-dose Cyclophosphamide (CYP; 300 mg/m²) (Johnson/ Mead, N.J.), cytokines such as γ-interferon, IL-2, or IL-12 or genes encoding proteins involved in immune helper functions, such as B-7.

[0304] The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization.

[0305] A second, booster injection, may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate MAbs.

[0306] For production of rabbit polyclonal antibodies, the animal can be bled through an ear vein or alternatively by cardiac puncture. The removed blood is allowed to coagulate and then centrifuged to separate serum components from whole cells and blood clots. The serum may be used as is for various applications or else the desired antibody fraction may be purified by well-known methods, such as affinity chromatography using another antibody, a peptide bound to a solid matrix, or by using, e.g., protein A or protein G chromatography.

[0307] MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified sGC protein, polypeptide, peptide or domain, be it a wild-type or mutant composition. The immunizing composition is administered in a manner effective to stimulate antibody producing cells.

[0308] The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, pp. 60-61), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.

[0309] The animals are injected with antigen, generally as described above. The antigen may be coupled to carrier molecules such as keyhole limpet hemocyanin if necessary. The antigen would typically be mixed with adjuvant, such as Freund's complete or incomplete adjuvant. Booster injections with the same antigen would occur at approximately two-week intervals.

[0310] Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible.

[0311] Often, a panel of animals will have been immunized and the spleen of an animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5×10⁷ to 2×10⁸ lymphocytes.

[0312] The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).

[0313] Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp. 65-66, 1986; Campbell, 1984). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp2 10-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bull for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.

[0314] One preferred murine myeloma cell is the NS-1 myeloma cell line (also termed P3-NS-1-Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.

[0315] Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion, though the proportion may vary from about 20:1 to about 1: 1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use of electrically induced fusion methods is also appropriate (Goding pp. 71-74, 1986).

[0316] Fusion procedures usually produce viable hybrids at low frequencies, about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.

[0317] The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells.

[0318] This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.

[0319] The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide MAbs. The cell lines may be exploited for MAb production in two basic ways. First, a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion (e.g., a syngeneic mouse). Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration. Second, the individual cell lines could be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.

[0320] MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography. Fragments of the monoclonal antibodies of the invention can be obtained from the monoclonal antibodies so produced by methods which include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer.

[0321] It is also contemplated that a molecular cloning approach may be used to generate monoclonals. For this, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the spleen of the immunized animal, and phagemids expressing appropriate antibodies are selected by panning using cells expressing the antigen and control cells. The advantages of this approach over conventional hybridoma techniques are that approximately 10⁴ times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination which further increases the chance of finding appropriate antibodies.

[0322] Alternatively, monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer, or by expression of full-length gene or of gene fragments in E. coli.

[0323] C. Antibody Conjugates

[0324] The present invention further provides antibodies against sGC proteinaceous molecules, generally of the monoclonal type, that are linked to one or more other agents to form an antibody conjugate. Any antibody of sufficient selectivity, specificity and affinity may be employed as the basis for an antibody conjugate. Such properties may be evaluated using conventional immunological screening methodology known to those of skill in the art.

[0325] Certain examples of antibody conjugates are those conjugates in which the antibody is linked to a detectable label. “Detectable labels” are compounds or elements that can be detected due to their specific functional properties, or chemical characteristics, the use of which allows the antibody to which they are attached to be detected, and further quantified if desired. Another such example is the formation of a conjugate comprising an antibody linked to a cytotoxic or anti-cellular agent, as may be termed “immunotoxins” (described in U.S. Pat. Nos. 5,686,072, 5,578,706, 4,792,447, 5,045,451, 4,664,911 and 5,767,072, each incorporated herein by reference).

[0326] Antibody conjugates are thus preferred for use as diagnostic agents. Antibody diagnostics generally fall within two classes, those for use in vitro diagnostics, such as in a variety of immunoassays, and those for use in vivo diagnostic protocols, generally known as “antibody-directed imaging”. Again, antibody-directed imaging is less preferred for use with this invention.

[0327] Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, e.g., U.S. Pat. Nos. 5,021,236 and 4,472,509, both incorporated herein by reference). Certain attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a DTPA attached to the antibody (U.S. Pat. No. 4,472,509). Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or period. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate.

[0328] In the case of paramagnetic ions, one might mention by way of example ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III).

[0329] In the case of radioactive isotopes for therapeutic and/or diagnostic application, one might mention astatine²¹¹, ¹⁴carbon, ⁵¹chromium, ³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷,¹⁵²Eu, galium⁶⁷ ,³hydrogen, iodine ^(123,) , iodine¹²⁵, iodine¹³¹, indium¹¹¹,⁵⁹iron, ³²phosphorus, rhenium¹⁸⁶, rhenium ¹⁸⁸, ⁷⁵selenium, ³⁵sulphur, technicium^(99m) and yttrium⁹⁰. ¹²⁵I is often being preferred for use in certain embodiments, and technicium⁹⁹m and indium¹¹¹are also often preferred due to their low energy and suitability for long range detection.

[0330] Radioactively labeled monoclonal antibodies of the present invention may be produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies according to the invention may be labeled with technetium-^(99m) by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column or by direct labeling techniques, e.g., by incubating pertechnate, a reducing agent such as SNCl₂, a buffer solution such as sodium-potassium phthalate solution, and the antibody. Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTPA) and ethylene diaminetetracetic acid (EDTA). Also contemplated for use are fluorescent labels, including rhodamine, fluorescein isothiocyanate and renographin.

[0331] The much preferred antibody conjugates of the present invention are those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase and glucose oxidase. Preferred secondary binding ligands are biotin and avidin or streptavidin compounds. The use of such labels is well known to those of skill in the art in light and is described, for example, in U.S. Pat. No. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incorporated herein by reference.

[0332] D. Immunodetection Methods

[0333] In still further embodiments, the present invention concerns immunodetection methods for binding, purifying, removing, quantifying or otherwise generally detecting biological components such as sGC proteinaceous components. The sGC antibodies prepared in accordance with the present invention may be employed to detect wild-type or mutant sGC proteins, polypeptides or peptides. As described throughout the present application, the use of wild-type or mutant sGC specific antibodies is contemplated. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Nakamura et al. (1987), incorporated herein by reference.

[0334] In general, the immunobinding methods include obtaining a sample suspected of containing an sGC protein, polypeptide or peptide, and contacting the sample with a first anti-sGC antibody in accordance with the present invention, as the case may be, under conditions effective to allow the formation of immunocomplexes.

[0335] These methods include methods for purifying wild-type or mutant sGC proteins, polypeptides or peptides as may be employed in purifying wild-type or mutant sGC proteins, polypeptides or peptides from patients′ samples or for purifying recombinantly expressed wild-type or mutant sGC proteins, polypeptides or peptides. In these instances, the antibody removes the antigenic wild-type or mutant sGC protein, polypeptide or peptide component from a sample. The antibody will preferably be linked to a solid support, such as in the form of a column matrix, and the sample suspected of containing the wild-type or mutant sGC protein antigenic component will be applied to the immobilized antibody. The unwanted components will be washed from the column, leaving the antigen immunocomplexed to the immobilized antibody, which wild-type or mutant sGC protein, polypeptide or peptide antigen is then collected by removing the wild-type or mutant sGC protein, polypeptide or peptide from the column.

[0336] The immunobinding methods also include methods for detecting or quantifying the amount of a wild-type or mutant sGC proteinaceous reactive component in a sample, which methods require the detection or quantification of any immune complexes formed during the binding process. Here, one would obtain a sample suspected of containing a wild-type or mutant sGC protein, polypeptide or peptide, and contact the sample with an antibody against wild-type or mutant sGC, and then detect or quantify the amount of immune complexes formed under the specific conditions.

[0337] In terms of antigen detection, the biological sample analyzed may be any sample that is suspected of containing a wild-type or mutant sGC proteinaceous molecule-specific antigen, such as a diseased urogenital tract tissue section, secretion or specimen, separated or purified forms of any of the above wild-type or mutant sGC proteinaceous-containing compositions.

[0338] Contacting the chosen biological sample with the antibody under conditions effective and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time lone enough for the antibodies to form immune complexes with, i.e., to bind to, any sGC antigens present. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.

[0339] In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological or enzymatic tags. U.S. Pat. Nos. concerning the use of such labels include 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated herein by reference. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody or a biotin/avidin ligand binding arrangement, as is known in the art.

[0340] The sGC antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined. Alternatively, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a “secondary” antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.

[0341] Further methods include the detection of primary immune complexes by a two step approach. A second binding ligand, such as an antibody, that has binding affinity for the antibody is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.

[0342] 1. ELISAs

[0343] As detailed above, immunoassays, in their most simple and direct sense, are binding assays. Certain preferred inumunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analyses, and the like may also be used.

[0344] In one exemplary ELISA, the anti-sGC antibodies of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the wild-type or mutant sGC antigen, such as a clinical sample, is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound wild-type or mutant sGC protein, polypeptide or peptide antigen may be detected. Detection is generally achieved by the addition of another anti-sGC antibody that is linked to a detectable label. This type of ELISA is a simple “sandwich ELISA”. Detection may also be achieved by the addition of a second anti-sGC antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

[0345] In another exemplary ELISA, the samples suspected of containing the wild-type or mutant sGC antigen are immobilized onto the well surface and then contacted with the anti-sGC antibodies of the invention. After binding and washing to remove non-specifically bound immune complexes, the bound anti-sGC antibodies are detected. Where the initial anti-sGC antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-sGC antibody, with the second antibody being linked to a detectable label.

[0346] Another ELISA in which the wild-type or mutant sGC proteins, polypeptides or peptides are immobilized, involves the use of antibody competition in the detection. In this ELISA, labeled antibodies against wild-type or mutant sGC protein, polypeptide or peptides are added to the wells, allowed to bind, and detected by means of their label. The amount of wild-type or mutant sGC antigen in an unknown sample is then determined by mixing the sample with the labeled antibodies against wild-type or mutant sGC before or during incubation with coated wells. The presence of wild-type or mutant sGC proteinaceous molecule in the sample acts to reduce the amount of antibody against wild-type or mutant sGC proteinaceous molecule available for binding to the well and thus reduces the ultimate signal. This is also appropriate for detecting antibodies against wild-type or mutant sGC protein, polypeptide or peptide in an unknown sample, where the unlabeled antibodies bind to the antigen-coated wells and also reduces the amount of antigen available to bind the labeled antibodies.

[0347] Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.

[0348] In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then “coated” with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein and solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

[0349] In ELISAs, it is probably more customary to use a secondary or tertiary detection means rather than a direct procedure. Thus, after binding of a proteinaceous molecule or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, or a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or third binding ligand.

[0350] “Under conditions effective to allow immune complex (antigen/antibody) formation” means that the conditions preferably include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.

[0351] The “suitable” conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25° C. to 27° C., or may be overnight at about 4° C. or so.

[0352] Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.

[0353] To provide a detecting means, the second or third antibody will have an associated label to allow detection. Preferably, this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the first or second immune complex with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).

[0354] After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS) and H₂O₂, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.

[0355] 2. Immunohistochemistry

[0356] The antibodies of the present invention may also be used in conjunction with both fresh-frozen and formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC). The method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and is well known to those of skill in the art (Brown etaL., 1990; Abbondanzo et al., 1990; Allred et al., 1990).

[0357] Briefly, frozen-sections may be prepared by rehydrating frozen “pulverized” tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and pelleting again by centrifugation; snap-freezing in −70° C. isopentane; cutting the plastic capsule and removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and cutting 25-50 serial sections.

[0358] Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and embedding the block in paraffin; and cutting up to 50 serial permanent sections.

[0359] VII. Diagnostics and Screens for Mammalian sGC

[0360] A. Diagnostics

[0361] As with the therapeutic methods of the present invention, the diagnostic methods are based upon the novel gene encoding sGC, which encode a protein that is predicted to have sGC activity. The diagnostic methods of the present invention generally involve determining either the type or the amount of a wild-type or mutant sGC proteinaceous molecule present within a biological sample from a patient suspected of having a disease associated with aberrant sGC activity. Irrespective of the actual role of sGC in the etiology of disease, it will be understood that the detection of a mutant form of sGC is likely to be diagnostic of a disease, such as those described herein, and that the detection of altered amounts of sGC, either at the mRNA or protein level, is also likely to have diagnostic implications, particularly where there is a reasonably significant difference in amounts.

[0362] The finding of a decreased amount of sGC in one, or preferably more, cancerous samples, in comparison to the amount within a sample from a control sample, will be indicative of the role of sGC in a particular disease. Following which, disease in others would be similarly diagnosed by detecting a decreased amount of sGC in a sample. The finding of a increased amount of sGC in one, or preferably more, patients, in comparison to the amount within a sample from a control subject, will be indicative of the role of the sGC in a particular disease. Following which, disease in others would be similarly diagnosed by detecting a increased amount of sGC in a sample.

[0363] The type or amount of sGC proteinaceous molecule present within a biological sample, such as a tissue sample, secretion, or body fluid, may be determined by means of a molecular biological assay to determine the level of a nucleic acid that encodes such an sGC proteinaceous molecule, or by means of an immunoassay to determine the level of the protein, polypeptide or peptide itself. Any of the foregoing nucleic acid detection methods or immunodetection methods may be employed as a diagnostic methods in the context of the present invention.

[0364] B. Modulators and Screening Assays

[0365] The present invention further comprises methods for identifying modulators of the function of sGC. These assays may comprise random screening of large libraries of candidate substances; alternatively, the assays may be used to focus on particular classes of compounds selected with an eye towards structural attributes that are believed to make them more likely to modulate the function of sGC.

[0366] To identify a sGC modulator, one generally will determine the function of sGC in the presence and absence of the candidate substance, a modulator defined as any substance that alters function. For example, a method generally comprises:

[0367] (a) providing a candidate modulator;

[0368] (b) admixing the candidate modulator with an isolated compound or cell, or a suitable experimental animal;

[0369] (c) measuring one or more characteristics of the compound, cell or animal in step (c); and

[0370] (d) comparing the characteristic measured in step (c) with the characteristic of the compound, cell or animal in the absence of said candidate modulator,

[0371] wherein a difference between the measured characteristics indicates that said candidate modulator is, indeed, a modulator of the compound, cell or animal.

[0372] Assays may be conducted in cell free systems, in isolated cells, or in organisms including transgenic animals.

[0373] It will, of course, be understood that all the screening methods of the present invention are useful in themselves notwithstanding the fact that effective candidates may not be found. The invention provides methods for screening for such candidates, not solely methods of finding them.

[0374] 1. Modulators

[0375] As used herein the term “candidate substance” refers to any molecule that may potentially inhibit or enhance sGC activity. The candidate substance may be a protein or fragment thereof, a small molecule, or even a nucleic acid molecule. Using lead compounds to help develop improved compounds is know as “rational drug design” and includes not only comparisons with know inhibitors and activators, but predictions relating to the structure of target molecules.

[0376] The goal of rational drug design is to produce structural analogs of biologically active polypeptides or target compounds. By creating such analogs, it is possible to fashion drugs, which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules. In one approach, one would generate a three-dimensional structure for a target molecule, or a fragment thereof. This could be accomplished by x-ray crystallography, computer modeling or by a combination of both approaches.

[0377] It also is possible to use antibodies to ascertain the structure of a target compound activator or inhibitor. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of anti-idiotype would be expected to be an analog of the original antigen. The anti-idiotype could then be used to identify and isolate peptides from banks of chemically- or biologically-produced peptides. Selected peptides would then serve as the pharmacore. Anti-idiotypes may be generated using the methods described herein for producing antibodies, using an antibody as the antigen.

[0378] On the other hand, one may simply acquire, from various commercial sources, small molecule libraries that are believed to meet the basic criteria for useful drugs in an effort to “brute force” the identification of useful compounds. Screening of such libraries, including combinatorially generated libraries (e.g., peptide libraries), is a rapid and efficient way to screen large number of related (and unrelated) compounds for activity. Combinatorial approaches also lend themselves to rapid evolution of potential drugs by the creation of second, third and fourth generation compounds modeled of active, but otherwise undesirable compounds.

[0379] Candidate compounds may include fragments or parts of naturally-occurring compounds, or may be found as active combinations of known compounds, which are otherwise inactive. It is proposed that compounds isolated from natural sources, such as animals, bacteria, fungi, plant sources, including leaves and bark, and marine samples may be assayed as candidates for the presence of potentially useful pharmaceutical agents. It will be understood that the pharmaceutical agents to be screened could also be derived or synthesized from chemical compositions or man-made compounds. Thus, it is understood that the candidate substance identified by the present invention may be peptide, polypeptide, polynucleotide, small molecule inhibitors or any other compounds that may be designed through rational drug design starting from known inhibitors or stimulators.

[0380] Other suitable modulators include antisense molecules, ribozymes, and antibodies (including single chain antibodies), each of which would be specific for the target molecule. Such compounds are described in greater detail elsewhere in this document. For example, an antisense molecule that bound to a translational or transcriptional start site, or splice junctions, would be ideal candidate inhibitors.

[0381] In addition to the modulating compounds initially identified, the inventors also contemplate that other sterically similar compounds may be formulated to mimic the key portions of the structure of the modulators. Such compounds, which may include peptidomimetics of peptide modulators, may be used in the same manner as the initial modulators.

[0382] An inhibitor according to the present invention may be one which exerts its inhibitory or activating effect upstream, downstream or directly on sGC. Regardless of the type of inhibitor or activator identified by the present screening methods, the effect of the inhibition or activator by such a compound results in altering sGC activity or expression as compared to that observed in the absence of the added candidate substance.

[0383] 2. In vitro Assays

[0384] A quick, inexpensive and easy assay to run is an in vitro assay. Such assays generally use isolated molecules, can be run quickly and in large numbers, thereby increasing the amount of information obtainable in a short period of time. A variety of vessels may be used to run the assays, including test tubes, plates, dishes and other surfaces such as dipsticks or beads.

[0385] One example of a cell free assay is a binding assay. While not directly addressing function, the ability of a modulator to bind to a target molecule in a specific fashion is strong evidence of a related biological effect. For example, binding of a molecule to a target may, in and of itself, be inhibitory, due to steric, allosteric or charge-charge interactions. The target may be either free in solution, fixed to a support, expressed in or on the surface of a cell. Either the target or the compound may be labeled, thereby permitting determining of binding. Usually, the target will be the labeled species, decreasing the chance that the labeling will interfere with or enhance binding. Competitive binding formats can be performed in which one of the agents is labeled, and one may measure the amount of free label versus bound label to determine the effect on binding.

[0386] A technique for high throughput screening of compounds is described in WO 84/03564. Large numbers of small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. Bound polypeptide is detected by various methods.

[0387] 3. In cyto Assays

[0388] The present invention also contemplates the screening of compounds for their ability to modulate sGC in cells. Various cell lines can be utilized for such screening assays, including cells specifically engineered for this purpose.

[0389] Depending on the assay, culture may be required. The cell is examined using any of a number of different physiologic assays. Alternatively, molecular analysis may be performed, for example, looking at protein expression, mRNA expression (including differential display of whole cell or polyA RNA) and others.

[0390] 4. In vivo Assays

[0391] In vivo assays involve the use of various animal models, including transgenic animals that have been engineered to have specific defects, or carry markers that can be used to measure the ability of a candidate substance to reach and effect different cells within the organism. Due to their size, ease of handling, and information on their physiology and genetic make-up, mice are a preferred embodiment, especially for transgenics. However, other animals are suitable as well, including rats, rabbits, hamsters, guinea pigs, gerbils, woodchucks, cats, dogs, sheep, goats, pigs, cows, horses and monkeys (including chimps, gibbons and baboons). Assays for modulators may be conducted using an animal model derived from any of these species.

[0392] In such assays, one or more candidate substances are administered to an animal, and the ability of the candidate substance(s) to alter one or more characteristics, as compared to a similar animal not treated with the candidate substance(s), identifies a modulator. The characteristics may be any of those discussed above with regard to the function of a particular compound (e.g., enzyme, receptor, hormone) or cell (e.g., growth, tumorigenicity, survival), or instead a broader indication such as behavior, anemia, immune response, etc.

[0393] The present invention provides methods of screening for a candidate substance that alter sGC activity or expression. In these embodiments, the present invention is directed to a method for determining the ability of a candidate substance to alter sGC activity or expression, generally including the steps of: administering a candidate substance to the animal; and determining the ability of the candidate substance to reduce one or more characteristics of sGC.

[0394] Treatment of these animals with test compounds will involve the administration of the compound, in an appropriate form, to the animal. Administration will be by any route that could be utilized for clinical or non-clinical purposes, including but not limited to oral, nasal, buccal, or even topical. Alternatively, administration may be by intratracheal instillation, bronchial instillation, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Specifically contemplated routes are systemic intravenous injection, regional administration via blood or lymph supply, or directly to an affected site.

[0395] Determining the effectiveness of a compound in vivo may involve a variety of different criteria. Also, measuring toxicity and dose response can be performed in animals in a more meaningful fashion than in in vitro or in cyto assays.

[0396] VIII. Pharmaceutical Compositions

[0397] Pharmaceutical compositions of the present invention comprise an effective amount of one or more sGC proteinaceous sequence, nucleic acid or antibody or additional agent dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of an pharmaceutical composition that contains at least one sGC proteinaceous sequence, nucleic acid or antibody or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[0398] As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0399] The sGC proteinaceous sequence, nucleic acid or antibody may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, rectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, intravesicularlly, mucosally, intrapericardially, orally, topically, locally, using aerosol, injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

[0400] The actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0401] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0. 1% of an active compound. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.

[0402] In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

[0403] The sGC proteinaceous sequence, nucleic acid or antibody may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.

[0404] In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.

[0405] In other embodiments, one may use eye drops, nasal solutions or sprays, aerosols or inhalants in the present invention. Such compositions are generally designed to be compatible with the target tissue type. In a non-limiting example, nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained. Thus, in preferred embodiments the aqueous nasal solutions usually are isotonic or slightly buffered to maintain a pH of about 5.5 to about 6.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, drugs, or appropriate drug stabilizers, if required, may be included in the formulation. For example, various commercial nasal preparations are known and include drugs such as antibiotics or antihistamines.

[0406] In certain embodiments the sGC proteinaceous sequence, nucleic acid or antibody is prepared for administration by such routes as oral ingestion. In these embodiments, the solid composition may comprise, for example, solutions, suspensions, emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatin capsules), sustained release formulations, buccal compositions, troches, elixirs, suspensions, syrups, wafers, or combinations thereof. Oral compositions may be incorporated directly with the food of the diet. Preferred carriers for oral administration comprise inert diluents, assimilable edible carriers or combinations thereof. In other aspects of the invention, the oral composition may be prepared as a syrup or elixir. A syrup or elixir, and may comprise, for example, at least one active agent, a sweetening agent, a preservative, a flavoring agent, a dye, a preservative, or combinations thereof.

[0407] In certain preferred embodiments an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, and combinations thereof. In certain embodiments, a composition may comprise one or more of the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.; or combinations thereof the foregoing. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both.

[0408] Additional formulations which are suitable for other modes of administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum, vagina or urethra. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.

[0409] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.

[0410] The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fingi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.

[0411] In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.

[0412] IX. Kits

[0413] Certain embodiments of the present invention concerns diagnostic or therapeutic kits. The components of the various kits may be stored in suitable container means. The container means will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the mammalian sGC proteinaceous molecule, nucleic acid, antibody or inhibitory formulation are placed, preferably, suitably allocated. The kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer or other diluent. The kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.

[0414] In one embodiment, a diagnostic kit may comprising sGC probes or primers for use with the nucleic acid detection methods. All the essential materials and reagents required for detecting sGC nucleic acid markers in a biological sample may be assembled together in a kit. This generally will comprise preselected primers for specific markers. Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (RT, Taq, etc.), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification.

[0415] Such kits generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each marker primer pair. Preferred pairs of primers for amplifying nucleic acids are selected to amplify the sequences specified in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6, or a complement thereof.

[0416] In another embodiment, such kits will comprise hybridization probes specific for sGC corresponding to the sequences specified in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6, or the complement thereof. Such kits generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each hybridization probe.

[0417] In other embodiments, the present invention concerns immunodetection kits for use with the immunodetection methods described above. As the sGC antibodies are generally used to detect wild-type or mutant sGC proteins, polypeptides or peptides, the antibodies will preferably be included in the kit. The immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to a wild-type or mutant sGC protein, polypeptide or peptide, and optionally, an immunodetection reagent and further optionally, a wild-type or mutant sGC protein, polypeptide or peptide.

[0418] In preferred embodiments, monoclonal antibodies will be used. In certain embodiments, the first antibody that binds to the wild-type or mutant sGC protein, polypeptide or peptide may be pre-bound to a solid support, such as a column matrix or well of a microtitre plate.

[0419] The immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with or linked to the given antibody. Detectable labels that are associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.

[0420] The kits may further comprise a suitably aliquoted composition of the wild-type or mutant sGC protein, polypeptide or polypeptide, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay. The kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit. The components of the kits may be packaged either in aqueous media or in lyophilized form.

[0421] Therapeutic kits of the present invention are kits comprising an sGC protein, polypeptide, peptide, biological functional equivalent, immunological fragment, domain, inhibitor, gene, vector, probe, primer, polynucleotide, nucleic acid, complement, antibody, or other sGC effector. Such kits will generally contain, in suitable container means, a pharmaceutically acceptable formulation of an sGC protein, polypeptide, peptide, biological functional equivalent, immunological fragment, domain, inhibitor, antibody, gene, polynucleotide, nucleic acid, complement, or vector expressing any of the foregoing in a pharmaceutically acceptable formulation. The kit may have a single container means, or it may have distinct container means for each compound.

[0422] When the components of the kit are provided in one or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. The sGC compositions may also be formulated into a syringeable composition. In which case, the container means may itself be a syringe, pipette, or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, or even applied to and mixed with the other components of the kit.

[0423] However, the components of the kit may be provided as dried powder(s). When reagents or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.

[0424] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antibody may be placed, and preferably, suitably aliquoted. Where wild-type or mutant sGC protein, polypeptide or peptide, or a second or third binding ligand or additional component is provided, the kit will also generally contain a second, third or other additional container into which this ligand or component may be placed. The kits of the present invention will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

[0425] Irrespective of the number or type of containers, the kits of the invention may also comprise, or be packaged with, an instrument for assisting with the injection/administration or placement of the ultimate sGC proteinaceous molecule or nucleic acid composition within the body of an animal. Such an instrument may be a syringe, pipette, forceps, or any such medically approved delivery vehicle.

[0426] X. Examples

[0427] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1

[0428] The structures of the genes encoding the α₁ and β₁ subunits of murine soluble guanylyl cyclase (sGC) were determined. Full-length cDNA's isolated from mouse lungs encoding the α₁, (2.5-kb) and β₁(3.3-kb) subunits are presented. The α₁. sGC gene is approximately 26.4 kb and contains 9 exons, while the β₁sGC gene spans 22 kb and has 14 exons. The positions of exon/intron boundaries and the sizes of introns for both genes are described. Comparison of mouse genomic organization with the Human Genome database predicted the exon/intron boundaries of the human genes and revealed that human and mouse α₁and β₁sGC genes have similar structures.

[0429] Both mouse genes are localized on the third chromosome, band 3E3-F1, and are separated by a fragment that is 2% of the chromosomal length. The 5′ untranscribed regions of α₁and β₁subunit genes were subcloned into luciferase reporter constructs and the functional analysis of promoter activity was performed in murine neuroblastoma NiE-115 cells. Results indicate that the 5′ untranscribed regions for both genes possess independent promoter activities and, together with the data on chromosomal localization, indicate independent regulation of both genes.

[0430] Abbreviations: sGC, soluble guanylyl cyclase; bp, base pairs; NO, nitric oxide; cAMP, cyclic adenosine monophosphate; FISH, fluorescence in situ hybridization; DAPI, 4′, 6-diamidino-2-phenylindole; CMV, cytomegalovirus.

[0431] Isolation of a eDNA clone for mouse sGCα₁subunit A mouse lung λ Triplex cDNA library (Clontech) was screened by hybridization using a 1.3-kb rat sGCOα₁cDNA fragment obtained by PCR using Taq polymerase (Gibco) and the oligonucleotide primers 5′-⁹¹TGCACTTCAGAGAACCTTG-3′ and 5′-⁵²⁰CTCCACCTTGTAGACATCCA-3′ (superscript indicates position of codon at which the primers start). Six positive clones were identified from approximately 1×10⁶ independent phage plaques. Positive clones were subsequently purified, sequenced bidirectionally for positive clone identification, and analyzed using DNASTAR software (DNASTAR, Inc., Madison, Wis.). Following analysis, the clone was defined as mouse (XI sGC and submitted to the NCBI database. This clone was used in all subsequent experiments and alignments described herein.

[0432] Isolation of genomic clones for mouse sGCα₁and β₁subunits. A bacterial artificial chromosomal (BAC) high-density membrane mouse library was purchased from Genome Systems (St. Louis, Mo.). The hybridization was performed overnight at 46° C. in standard hybridization solution (Sambrook et al.,1989). A random primer-labeled α³²P-dCTP-labeled cDNA fragment (0.9 Kb) for the β₁sGC probe was generated by RT-PCR from a total RNA preparation from murine neuroblastoma N1E-115 cells, using the oligonucleotides 5 ′-−3GACACCATGTACGGTTTCGTG-3′ and 5′-²⁴³CCCTTCCTTGCTTCTCAGTAC-3′ (superscript indicates the base pairs upstream of the start codon or the position of the codon at which the primers start).

[0433] The membranes were then re-hybridized with an α₁sGC cDNA probe (1.3 kb containing coding sequence) using the same conditions. Positive BAC clones were identified using the manufacturer's procedure and purchased from Genome Systems (St. Louis, Mo.). BAC plasmid purification kit (Clontech) was used for BAC DNA isolation from bacterial culture. BAC DNA was subjected to restriction and Southern blot hybridization analysis ((Sambrook et al.,1989)) using the same hybridization probes to confirm isolation of positive clones (data not shown).

[0434] Determination of boundaries and sizes of introns. Based on the α₁and β₁ sGC cDNA sequences, sequencing oligonucleotide primers were designed to determine the genomic structure of each subunit. All sequencing analyses were performed at the Molecular Core Sequencing Facility at the University of Texas-Houston Medical School on an ABI Prism 377 DNA sequencer with the DigDye Terminator cycle sequencing kit (Applied Biosystems, Calif.). Primers positioned in the exons of both subunits were used to determine the intron sizes by PCR with Pfu-Turbo DNA polymerase (Stratagene) from BAC DNA templates. PCR conditions were: melting step at 95° C. for 1 min, primer annealing at 55° C. for 1 min, extension step at 72° C. for 3 min, repeated for 35 cycles. PCR products were separated by electrophoresis on 1% agarose gels.

[0435] 3′- Rapid amplification of cDNA end (3′-RACE) of mouse sGC,β₁subunit. Poly (A)⁺ RNA was purified from lung tissue of CD57 mice using an MRNA extraction kit (Dynal). Determination of the 3′ end of β₁sGC MRNA was performed using a SMART RACE cDNA Amplification kit (Clontech). In brief, the first-strand cDNA synthesis was achieved by incubating the poly(A)⁺ RNA with a 3′cDNA-specific primer and Superscript Reverse Transcriptase (Gibco) for 1.5 hrs at 42° C. Next a “touchdown” PCR reaction to amplify the fragment was executed using an oligonucleotide

[0436] 5′-²⁵⁸CTGCTACAAGCATTGCCTAGACGGACG-3′ (superscript indicates the base pairs downstream of the stop codon where the primer starts), specific to the 3′ end of the published β₁sGC sequence, and the universal primer mixture that recognizes the modified 3′-end of cDNA. PCR conditions were as suggested by the manufacturer (Clonthech). PCR products were subdloned into pCR 2.1-Topo vector (Invitrogen) and both strands were sequenced for verification.

[0437] Chromosomal localization of mouse sGCα₁and β_(1 subunits. Chromosomal localization of α) ₁and β₁sGC genes was performed by Genome Systems (St. Louis, Mo.) using fluorescence in situ hybridization (FISH). Briefly, purified BAC DNA for each clone, containing the genomic sequence of α₁and β₁sGC, was labeled with digoxigenin dUTP by nick translation. The labeled probe was combined with sheared mouse DNA and hybridized to normal metaphase chromosomes derived from mouse embryo fibroblast cells in a solution containing 50% formamide, 10 % dextran sulfate, and 2×SSC. The hybridization was detected using fluorescent antidigoxigenin antibodies followed by counterstaining with DAPI. In addition, a probe specific for the telomeric region of chromosome 3 was co-hybridized with each clone to verify specific labeling of the telomere to chromosome 3. Specific measurements identifying the hybridization signal between the heterochromatic-euchromatic boundary to the telomere of chromosome 3 indicated the band location of each clone on mouse chromosome 3.

[0438] Cloning of luciferase plasmid constructs. In order to create the plasmid constructs containing the 5′-upstream regions of α₁and β₁sGC extended to the first identified exon for each gene, DNA fragments were obtained by PCR using the specific genomic clones as templates and Pfu Turbo DNA Polymerase (Stratagene). Positive strand oligonucleotide primers for each construct were:

[0439] 1.6 kb -α₁5′-¹⁹⁰¹GTCAGTGTCAGACCTGAAGATGCTG-3′ and

[0440] 1.4 kb -β₁5 ′-⁻¹⁵²⁸CTCTCTGTGTGTGAGAGAGAG-3′ (superscript indicates the base pairs upstream of the start codon). Each of these positive strand oligonucleotide primers contained a Kpn I restriction site linker sequence at the 5′ end. The negative strand primers were:

[0441] 5′-⁻¹⁰⁴CATGATGCGATCACAGGAGGC-3′for the α₁, construct and

[0442] 5′-⁻¹⁰⁵CGCCCGGAGCCTAGGAAGCAG-3′ for the β₁construct (superscript indicates the base pairs upstream of the start codon). Each of the negative strand primers contained a Bgl II restriction site linker sequence at the 5′ end. After restriction digestion of the ends, the PCR fragments were directionally cloned into the luciferase reporter vector pGL3-Basic (Promega) between the Kpn I and Bgl II restriction sites upstream of the luciferase gene.

[0443] Transfection and detection of luciferase activity. N1E-115 mouse neuroblastoma cells were maintained in DMEM with 4mM L-glutamine, 4.5 g/L glucose, 1% penicylin-streptomicyn mixture and 10% fetal bovine serum (Hyclone). Cells were transiently transfected with each (α₁and β₁ sGC) luciferase plasmid construct using Fugene-6 transfection reagent (Roche). Cultures were incubated in the presence of Fugene and DNA (1 μg) for 48 hr, and assayed for luciferase activity using a luciferase reporter assay (Promega). Cells were co-transfected with a β-galactosidase construct (CMV-β-gal, ⅕ of the concentration of sGC constructs) and assayed for β-gal activity in the N1E cell lysates to normalize the transfection efficiency between cell groups (not shown).

RESULTS:

[0444] Cloning of the mouse α₁ sGC subunit cDNA. The cDNA for mouse α₁ subunit of sGC was not previously isolated and reported. Described herein, 6 clones were isolated by screening a mouse cDNA library (Clontech) using a rat cDNA sequence as a probe. The clone containing the longest insertion was sequenced and analyzed for the presence of the open reading frame (ORF) encoding the α₁ sGC subunit. The sequence comparison of isolated cDNA demonstrated 93.3% and 83.9% homology with rat and human α₁ cDNA, respectively, confirming that the isolated clone indeed encodes mouse α₁ sGC. The sequence was submitted to the NCBI database.

[0445] Isolation of 3′ endfragment of β₁ sGC cDNA. The NCBI database contains a 2.3-kb cDNA sequence for mouse β₁ sGC (accession N AF020339). Northern analysis of mouse lung total RNA performed in our laboratory showed a 4-kb transcript for β₁ sGC (data not shown). To find the missing portion of the mRNA for β₁ sGC, a 3′-RACE analysis was performed on the mouse lung mRNA prepararation. The first cDNA strand was generated using a primer located upstream of the known 3′ end of mouse cDNA and the oligo-dT adaptor primer. A 1-kb fragment was successfully isolated. Sequence analysis of this fragment indicated that it contained the expected 70-bp region identical to the known mouse 3′ end of β₁ cDNA followed by a 956-bp novel sequence containing a conservative consensus for the polyadenylation signal and polyA stretch. The sequence was highly homologous to the rat 3′ end of β₁ cDNA (data not shown). This allowed us to conclude that the complete 3′-UTR for β₁ sGC cDNA had been isolated. The full CDNA sequence of mouse β₁ sGC subunit was submited to NCBI database.

[0446] Genomic organization of mouse α₁ and β₁ sGC genes. Three overlapping BAC clones were isolated for each of the sGC genes by separate screening of a BAC mouse genomic library (Genome Systems, Inc.) utilizing probes containing a 1.3-kb fragment of the coding sequence for mouse α₁ sGC and a 0.9-kb N-terminal fragment of the coding mouse β₁ sGC sequence. Southern analysis of isolated clones with probes specific for the 5′ and 3′ cDNA fragments for both sGC subunits confirmed that at least two out of three clones for each subunit contained a genomic fragment that hybridized with both 5′ and 3′ probes from the α₁ and β₁ cDNA's ( data not shown ). Genomic sequences isolated during screening have 99% and 100% homology in coding regions with the murine α₁ and β₁ cDNAs, respectively, confirming successful isolation of the genes for these two isoforms and subunits. Comparison of the coding sequences for the α₁ sGC subunit gene with previously cloned cDNA revealed seven mismatches in codons 49 (TAC→GAC), 52 (GAG→GAA), 319 (AAC→AGC), 343 (AAC→AAT), 445 (GAA→GAG), 487 (ATC→ACC) and 690 (GTA→ATA). Out of the seven codons, four of the replacements (49, 319, 487, 690) introduce different amino acid residues in the final protein sequence. The source for the BAC genomic library (Genomic Systems, Inc.) utilized in the analyses differed from that of the cDNA library (Clontech), indicating that the inconsistency in these sequences reflect DNA polymorphism between different strains of mice (i.e., 129/SvJ I vs. 200 BALB/c, respectively).

[0447] The positions of the exon/intron boundaries were identified by sequencing using oligonucletide primers located within the coding sequences of each gene (Table 6). TABLE 6 Exon-intron splice junctions of the α1 and β1 sGC genes. splice splice donor* size of intron (kb) acceptor α1 sGC INTRON 1  CAT⁻¹⁰³G/GTGGGTTCGCTCAGC >2.0 TCCACTGCTCATAG/GT GCT INTRON 2  CCA⁸⁵GAG/GTGAGTGTTCTCCCT 5.5 CTTTTTCTTTCCAG/TGT GAG INTRON 3  AAC¹⁰⁶AG/GTAAGCTAAGTTACC 2.2 TTAATTATTCCCAG/G AAA INTRON 4  GCA¹²⁶G/GTAATAAATAAAACT 1.9 CTGTGTGCTTGCAG/GTG CCC INTRON 5  TCA³⁶²AGG/GTAAGGAAAATGTAA 3.0 CCTTTCCTTTGCAG/GTT ATG INTRON 6  TAG⁵²⁴AAG/GTAGGGAAGGTGGAA 4.2 TATATTGTATGTAG/GTG GAG INTRON 7  ATC⁵⁷²AAG/GTA AGGCCGTGACT 4.4 TTGTTTTGCCTTCAG/ATG CGA INTRON 8  TAG⁶²⁴AG/GTATGGATGGCACTA 2.8 TAAATTGTTCTCAG/G TTA β1 sGC Intron 1  acc¹atg/GTGAGTGCTGTCAG 0.5 TCTCTGCCCTTCAG/tac ggt Intron 2  atc²⁶aa/GTAAGTGAACAGCC 2.5 TCCATTTTCTTTCAG/a aaa Intron 3  ctc⁶⁰a/GTAGGTTGAAAAC 2.4 CATCTACAAAACAG/ac ctc Intron 4  tttg⁹⁸cag/GTGAGATGTTCGAG 0.8 CTGCTGCACTACAG/aac ctc Intron 5  atg¹⁶⁴aag/GTAGTGTTCACCCG 1.1 CCATTGACATCTAG/gtg att Intron 6  ccc²⁴¹cag/GTAAAATGCACAG 1.1 TTTCTGTGTCTTAG/ctc cag Intron 7  agc²⁸⁰aag/GTAAGCAAGAACC 1.0 CTTTCCTGTTTAAG/gaa ggg Intron 8  cca³²⁵ag/GTAACAACTTTTAA 3.0 CTCTGTGTGACAG/t gtg Intron 9  gac³⁹¹ac/GTAAGCAAGGGAG 1.5 CTAATTCCCACAG/a ttg Intron 10 tac⁴⁷⁰aag/GCAAGTCTTCATGG 2.5 TGTGTCACCCTAG/gtg gaa Intron 11 gtt⁵¹⁷cag/GTGAGTAAATAAAT 0.4 CTTTGCTTCTGCAG/ata aca Intron 12 tac⁵⁶⁹ag/GTGAGGAGGGAAAT 0.3 CTCATGACTTTCAG/g tgt Intron 13 acg⁶¹¹gag/GTATGGCTCATTAG 1.1 TCGACCCATTTAAG/gaa aca

[0448] Intron sizes were estimated using PCR (see Table 6) and for introns 1, 2, 10, 11, 12 of β₁ sGC by complete sequencing. While the size of intron 1 for sGCα₁ was not determined, partial sequencing indicates it is more than 2 Kb.

[0449] The α₁ sGC gene encompasses at least 26.4 kb and includes 9 exons and 8 introns, while the β₁ sGC gene contains 14 exons and 13 introns and spans 22 kb. Start codons are positioned in the second and first exons of the α₁ and β₁ genes, respectively. The GT/AG donor/acceptor consensus was maintained in all introns for both genes, except for intron 10 in β₁ sGC, where the donor site was GC. The sequences that flank the exon/intron boundaries in the α₁ and β₁ sGC genes are presented in Table 1.

[0450] Chromosomal localization. BAC clones containing α₁ and β₁ genes were used for chromosomal localization by FISH analysis. DNA from BAC clones containing genomic regions of α₁ and β₁ sGC genes was labeled with digoxigenin dUTP and hybridized to normal metaphase chromosomes derived from mouse embryo fibroblast cells. A total of 80 metaphase cells were analysed for each genomic clone with 71 and 72 chromosomes exhibiting specific labeling for α₁ and β₁ sGC genes, respectively. Both genes co-localized to mouse chromosome 3. The α₁ sGC gene positioned at 44% and PI sGC at 46% of the distance from the heterochromatic-euchromatic boundary to the telomere of chromosome 3, which corresponds to band 3E3-F1.

[0451] Analysis of the promoter activity of 5′ regions for α₁ and β₁ sGC genes in the N1E-115 cell line. Murine neuroblastoma N1E-115 cells were selected for promoter analysis as host cells since expression of sGC was shown in this cell line (34). 1.6-kb and 1.4-kb of the 5′-flanking regions extended until the first identified exons of the α₁ and β₁ sGC subunit genes were subdloned upstream of the luciferase gene of the pGL3-Basic luciferase reporter vector (Promega). The constructs were transiently transfected in N1E-115 cells. Luciferase activity generated by each of the constructs was compared with the activity of the promotorless pGL3-basic plasmid. The upstream regions of the α₁ and β₁ sGC genes demonstrated different transcriptional activity in N1E-115 cells. Values were normalized using β-galactosidase construct (CMV-β-gal ) cotransfections and the total protein concentration of each group. The values obtained represented the normalized means ±S.D. of three different experiments. The relative level of luciferase activity for the β₁ sGC construct was 4.6-fold higher when compared with the control, while the α₁ sGC construct activity was only 2-fold higher than the control promoterless plasmid.

[0452] Prediction of the organization of the human sGC genes. cDNA's for mouse and human α₁ and β₁ subunits were compared with the Human Genome Database. A clone was identified containing putative genomic regions for both human α₁ and β₁ subunits (clone AC021433), which is ascribed to chromosome 2. Exons 3-9 of the human α₁ gene and all exons of the human β₁ gene were identified in this genomic fragment. The comparison of predicted exon-intron boundaries and donor/acceptor sites from the human genes with those of the mouse genes revealed that they are identical in both species with the exception of intron 4 (donor and acceptor sites) and intron 9 (acceptor site) of the β₁ subunit.

DISCUSSION

[0453] The mouse α₁ and β₁ sGC genes map to the third chromosome. However, they are separated from each other by an extended region comprising 2% of the total chromosomal length. This finding excludes the possibility of tandem organization and directly coordinated transcription as proposed for the fish genes. This conclusion is supported by the independent ability of the 5′-flanking regions of the α₁ and β₁ sGC genes to drive transcription. However, the trans-coordinated transcriptional regulation by the same factors is possible.

[0454] The rat α₁ andβ₁ genes map to chromosome 2 and are closely linked to the particulate guanylyl cyclase isoform locus (GC-A) and quantitative trait locus (QTL) which have been associated with salt-sensitive hypertension in Dahl rats (Azam, et al., 1998). Thus far, no direct connection of the genomic loci identified for α₁ and β₁ sGC to hypertension in mouse or human has been demonstrated (Danziger, et al., 2000). Here it is shown that both mouse genes co-localize on chromosome 3 in the area corresponding to band 3E3-F1. The α₁ and β₁ subunits are co-localized with Muc1, Pk1r, Ntrk1, CD1 and Fcfr1 loci located at 44.8-46.5 positions of chromosome 3. It is contemplated that mutations in these genes contributes to these diseases.

[0455] Probing of the Human Genome database with the mouse and human cDNA sequences identified the clone AC021433 which contains 8 exons of the human α₁ gene and 14 exons of the human β₁ gene. This fragment is ascribed to human chromosome two. Although this fragment is missing the first two exons of the α₁ gene this is the most probable candidate for the locus of human the α₁ and β₁ genes. In a previous report (Giuili, et al., 1993), α₃ and β₃ subunits of human sGC (later confirmed to be α₁ and β₁ sGC genes (Zabel, et al., 1998) were colocalized to human chromosome 4 at q31.1 -q33, what represents some discrepancy with recent results. However, previously reported mapping of the chromosomal position for α₁ and β₁ of sGC were made using cDNA for corresponding isoforms as probes for the localization (Giuili, et al., 1993). Considering the high level of homology between coding regions of both sGC subunits in various isoforms and, in addition, the existence of regions of extensive homologies (in the catalytic domain, for example) to the particulate guanylyl cyclase family, the use of cDNA for chromosomal localization could result in ambiguities.

[0456] The transcriptional regulation of the expression of sGC has not been previously examined. Recently, evidence to support altered expression of mRNA of sGC subunits has emerged. In primary rat pulmonary artery smooth muscle cells, prolonged NO treatment leads to decreased NO-stimulated sGC activity and mRNA levels (Filippov et al. 1997). sGC levels rise in unborn rat pulmonary artery, beginning at approximately 20 days of gestation and mRNA, protein, and activity remain elevated at least 8 days following birth (Bloch et al., 1997). Decreased rates of sGC transcription have also been indicated in other models following NO treatment and administration of cAMP-elevating agents (24;25). Furthermore, nerve growth factor administration to rat PC-12 cells results in decreased steady state levels of sGC α₁ and β₁ mRNA.

[0457] It was found by the inventors that estrogen treatment decreases α₁ and β₁ sGC mRNA levels in rat uterus. The precise mechanisms underlying these effects on sGC in specific tissues are largely unknown. The activity of putative promoter regions demonstrate different transcriptional activity for both subunits, demonstrating the potential for finely tuned regulation.

[0458] All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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1 15 1 2430 DNA Mus musculus CDS (286)..(2361) 1 gagaagtggg agggactcag agccgcgggt ttctcacaca ccgccttcta ggcagccctc 60 ctccagtgcc tgccagccgg accggacccc aaggcgaaga gcagcagtgc acagcctggg 120 gagccagcgg agcaaagaca cctttggccc gatgcccctg gcctcctgtg atcgcatcat 180 ggtgctgggt cactcctgtc cttgagtcag tagaagcaga tcttcatcag tccacatcaa 240 caccagctag tcagaaggaa gccactgcca agctccagga acacc atg ttc tgc agg 297 Met Phe Cys Arg 1 aag ttc aag gat ctc aag atc aca ggg gag tgt cct ttc tcc tta ctg 345 Lys Phe Lys Asp Leu Lys Ile Thr Gly Glu Cys Pro Phe Ser Leu Leu 5 10 15 20 gcc cct ggt cag gtt cct aag gag cca aca gag gag gtg gct gga ggc 393 Ala Pro Gly Gln Val Pro Lys Glu Pro Thr Glu Glu Val Ala Gly Gly 25 30 35 tct gag ggc tgc cag gct act ctg ccc atc tgc cag tac ttt cct gag 441 Ser Glu Gly Cys Gln Ala Thr Leu Pro Ile Cys Gln Tyr Phe Pro Glu 40 45 50 aag aat gca gaa ggg agt ctc ccc caa aga aag aca agc cgc aac aga 489 Lys Asn Ala Glu Gly Ser Leu Pro Gln Arg Lys Thr Ser Arg Asn Arg 55 60 65 gtc tac ctg cac acc ctg gca gag agt att tgc aag ctc atc ttc cca 537 Val Tyr Leu His Thr Leu Ala Glu Ser Ile Cys Lys Leu Ile Phe Pro 70 75 80 gag tgt gag cga ctg aac ctt gca ctt cag aga acc ttg gca aag cat 585 Glu Cys Glu Arg Leu Asn Leu Ala Leu Gln Arg Thr Leu Ala Lys His 85 90 95 100 aaa ata gaa gaa aac agg aaa tct tca gaa aaa gaa gac ctt gaa aaa 633 Lys Ile Glu Glu Asn Arg Lys Ser Ser Glu Lys Glu Asp Leu Glu Lys 105 110 115 ata atc gca gaa gaa gca att gca gca ggt gcc cca gtg gag gcg ctc 681 Ile Ile Ala Glu Glu Ala Ile Ala Ala Gly Ala Pro Val Glu Ala Leu 120 125 130 aaa gac tct ctg ggc gag gag ctg ttc aag atc tgc tat gag gaa gat 729 Lys Asp Ser Leu Gly Glu Glu Leu Phe Lys Ile Cys Tyr Glu Glu Asp 135 140 145 gag cac att ttg ggc gtg gtt ggc ggc acc ctg aag gac ttt cta aat 777 Glu His Ile Leu Gly Val Val Gly Gly Thr Leu Lys Asp Phe Leu Asn 150 155 160 agc ttc agc acg ctc ctc aag cag agc agc cac tgc caa gag gcg gag 825 Ser Phe Ser Thr Leu Leu Lys Gln Ser Ser His Cys Gln Glu Ala Glu 165 170 175 180 agg cgg gga cga ctg gaa gat gcc tcc atc tta tgc ctg gac aag gac 873 Arg Arg Gly Arg Leu Glu Asp Ala Ser Ile Leu Cys Leu Asp Lys Asp 185 190 195 cag gac ttt cta aat gtt tac tac ttc ttc ccg aag aga acc aca gcc 921 Gln Asp Phe Leu Asn Val Tyr Tyr Phe Phe Pro Lys Arg Thr Thr Ala 200 205 210 ctg ctt ctc cct ggt atc att aaa gcg gct gct cgc ata ctg tac gaa 969 Leu Leu Leu Pro Gly Ile Ile Lys Ala Ala Ala Arg Ile Leu Tyr Glu 215 220 225 agc cac gtg gag gtg tcc ctg atg cct ccc tgc ttc cga agt gac tgt 1017 Ser His Val Glu Val Ser Leu Met Pro Pro Cys Phe Arg Ser Asp Cys 230 235 240 acc gag ttt gtg aac cag ccc tat ttg ctc tac tcc gtt cat gtg aag 1065 Thr Glu Phe Val Asn Gln Pro Tyr Leu Leu Tyr Ser Val His Val Lys 245 250 255 260 agc acc aag ccg tcc ctg tcc cca ggc aag ccc cag tcc tcg cta gtg 1113 Ser Thr Lys Pro Ser Leu Ser Pro Gly Lys Pro Gln Ser Ser Leu Val 265 270 275 atc ccc gct tcg ctc ttc tgc aag act ttc ccg ttc cat ttc atg ctg 1161 Ile Pro Ala Ser Leu Phe Cys Lys Thr Phe Pro Phe His Phe Met Leu 280 285 290 gac cga gac ctg gcc atc ctg cag ctg ggt aac ggc atc aga agg ctg 1209 Asp Arg Asp Leu Ala Ile Leu Gln Leu Gly Asn Gly Ile Arg Arg Leu 295 300 305 gtg aac aag agg gac ttc caa ggg aag ccc aac ttt gaa gag ttc ttt 1257 Val Asn Lys Arg Asp Phe Gln Gly Lys Pro Asn Phe Glu Glu Phe Phe 310 315 320 gaa att cta act ccc aaa atc aac cag aca ttt agt ggc atc atg aca 1305 Glu Ile Leu Thr Pro Lys Ile Asn Gln Thr Phe Ser Gly Ile Met Thr 325 330 335 340 atg ttg aac atg cag ttt gtc atc cgg gtg agg aga tgg gat aac tcg 1353 Met Leu Asn Met Gln Phe Val Ile Arg Val Arg Arg Trp Asp Asn Ser 345 350 355 gtg aag aaa tcg tca agg gtt atg gat ctc aaa ggt caa atg atc tac 1401 Val Lys Lys Ser Ser Arg Val Met Asp Leu Lys Gly Gln Met Ile Tyr 360 365 370 atc gtt gaa tcc agt gcc atc ttg ttc tta ggg tca cca tgt gtg gac 1449 Ile Val Glu Ser Ser Ala Ile Leu Phe Leu Gly Ser Pro Cys Val Asp 375 380 385 agg ctg gaa gat ttc aca gga cgg ggg ctc tat ctg tcc gac atc cca 1497 Arg Leu Glu Asp Phe Thr Gly Arg Gly Leu Tyr Leu Ser Asp Ile Pro 390 395 400 att cat aac gcc ctg agg gat gtt gtc ttg ata ggg gag cag gca cgg 1545 Ile His Asn Ala Leu Arg Asp Val Val Leu Ile Gly Glu Gln Ala Arg 405 410 415 420 gct caa gat ggc ctc aag aag agg ttg ggg aag ctg aag gca acc ctg 1593 Ala Gln Asp Gly Leu Lys Lys Arg Leu Gly Lys Leu Lys Ala Thr Leu 425 430 435 gag cat gcc cac caa gcc ctg gag gaa gag aag aag agg aca gtg gat 1641 Glu His Ala His Gln Ala Leu Glu Glu Glu Lys Lys Arg Thr Val Asp 440 445 450 ctg ctg tgc tct atc ttc ccc tct gag gtt gct cag cag ctg tgg caa 1689 Leu Leu Cys Ser Ile Phe Pro Ser Glu Val Ala Gln Gln Leu Trp Gln 455 460 465 gga caa att gtg caa gcc aag aaa ttc agc gag gtc acc atg ctt ttc 1737 Gly Gln Ile Val Gln Ala Lys Lys Phe Ser Glu Val Thr Met Leu Phe 470 475 480 tca gat atc gta ggg ttc act gct atc tgc tct cag tgt tca cct ctg 1785 Ser Asp Ile Val Gly Phe Thr Ala Ile Cys Ser Gln Cys Ser Pro Leu 485 490 495 500 cag gtc atc acg atg ctc aac gct ctc tac act cgc ttt gac cag cag 1833 Gln Val Ile Thr Met Leu Asn Ala Leu Tyr Thr Arg Phe Asp Gln Gln 505 510 515 tgt gga gag ctg gat gtc tac aag gtg gag acc atc ggg gat gca tat 1881 Cys Gly Glu Leu Asp Val Tyr Lys Val Glu Thr Ile Gly Asp Ala Tyr 520 525 530 tgt gtg gca ggt gga ttg cac aga gag agt gac acc cat gct gtc cag 1929 Cys Val Ala Gly Gly Leu His Arg Glu Ser Asp Thr His Ala Val Gln 535 540 545 ata gca ctg atg gcc ctg aag atg atg gag ctc tcc aat gag gtc atg 1977 Ile Ala Leu Met Ala Leu Lys Met Met Glu Leu Ser Asn Glu Val Met 550 555 560 tct ccc cac gga gaa cct atc aag atg cga att gga cta cat tct gga 2025 Ser Pro His Gly Glu Pro Ile Lys Met Arg Ile Gly Leu His Ser Gly 565 570 575 580 tca gtg ttt gct gga gtt gtc gga gtg aag atg ccc cgg tat tgc ctg 2073 Ser Val Phe Ala Gly Val Val Gly Val Lys Met Pro Arg Tyr Cys Leu 585 590 595 ttt gga aac aat gtc act ctg gct aac aaa ttt gaa tcc tgc agt gtg 2121 Phe Gly Asn Asn Val Thr Leu Ala Asn Lys Phe Glu Ser Cys Ser Val 600 605 610 cct cgg aaa atc aat gtc agc ccc acc aca tac agg tta ctc aaa gac 2169 Pro Arg Lys Ile Asn Val Ser Pro Thr Thr Tyr Arg Leu Leu Lys Asp 615 620 625 tgt cct ggc ttt gtg ttc acc ccg aga tca agg gag gag ctt cca cca 2217 Cys Pro Gly Phe Val Phe Thr Pro Arg Ser Arg Glu Glu Leu Pro Pro 630 635 640 aac ttc cct agt gac att cct ggg atc tgt cac ttt ctg gat gct tat 2265 Asn Phe Pro Ser Asp Ile Pro Gly Ile Cys His Phe Leu Asp Ala Tyr 645 650 655 660 cac cat caa gga cct aat tcc aaa cca tgg ttc cag gat aaa gat gtg 2313 His His Gln Gly Pro Asn Ser Lys Pro Trp Phe Gln Asp Lys Asp Val 665 670 675 gaa gat gga aac gcc aac ttc tta ggc aaa gcg tca ggg gta gat tag 2361 Glu Asp Gly Asn Ala Asn Phe Leu Gly Lys Ala Ser Gly Val Asp 680 685 690 tgagccacat gctcttatgt ttgatgcctt tgaaggtgtg cagaacctct gtgttgacct 2421 taggattac 2430 2 691 PRT Mus musculus 2 Met Phe Cys Arg Lys Phe Lys Asp Leu Lys Ile Thr Gly Glu Cys Pro 1 5 10 15 Phe Ser Leu Leu Ala Pro Gly Gln Val Pro Lys Glu Pro Thr Glu Glu 20 25 30 Val Ala Gly Gly Ser Glu Gly Cys Gln Ala Thr Leu Pro Ile Cys Gln 35 40 45 Tyr Phe Pro Glu Lys Asn Ala Glu Gly Ser Leu Pro Gln Arg Lys Thr 50 55 60 Ser Arg Asn Arg Val Tyr Leu His Thr Leu Ala Glu Ser Ile Cys Lys 65 70 75 80 Leu Ile Phe Pro Glu Cys Glu Arg Leu Asn Leu Ala Leu Gln Arg Thr 85 90 95 Leu Ala Lys His Lys Ile Glu Glu Asn Arg Lys Ser Ser Glu Lys Glu 100 105 110 Asp Leu Glu Lys Ile Ile Ala Glu Glu Ala Ile Ala Ala Gly Ala Pro 115 120 125 Val Glu Ala Leu Lys Asp Ser Leu Gly Glu Glu Leu Phe Lys Ile Cys 130 135 140 Tyr Glu Glu Asp Glu His Ile Leu Gly Val Val Gly Gly Thr Leu Lys 145 150 155 160 Asp Phe Leu Asn Ser Phe Ser Thr Leu Leu Lys Gln Ser Ser His Cys 165 170 175 Gln Glu Ala Glu Arg Arg Gly Arg Leu Glu Asp Ala Ser Ile Leu Cys 180 185 190 Leu Asp Lys Asp Gln Asp Phe Leu Asn Val Tyr Tyr Phe Phe Pro Lys 195 200 205 Arg Thr Thr Ala Leu Leu Leu Pro Gly Ile Ile Lys Ala Ala Ala Arg 210 215 220 Ile Leu Tyr Glu Ser His Val Glu Val Ser Leu Met Pro Pro Cys Phe 225 230 235 240 Arg Ser Asp Cys Thr Glu Phe Val Asn Gln Pro Tyr Leu Leu Tyr Ser 245 250 255 Val His Val Lys Ser Thr Lys Pro Ser Leu Ser Pro Gly Lys Pro Gln 260 265 270 Ser Ser Leu Val Ile Pro Ala Ser Leu Phe Cys Lys Thr Phe Pro Phe 275 280 285 His Phe Met Leu Asp Arg Asp Leu Ala Ile Leu Gln Leu Gly Asn Gly 290 295 300 Ile Arg Arg Leu Val Asn Lys Arg Asp Phe Gln Gly Lys Pro Asn Phe 305 310 315 320 Glu Glu Phe Phe Glu Ile Leu Thr Pro Lys Ile Asn Gln Thr Phe Ser 325 330 335 Gly Ile Met Thr Met Leu Asn Met Gln Phe Val Ile Arg Val Arg Arg 340 345 350 Trp Asp Asn Ser Val Lys Lys Ser Ser Arg Val Met Asp Leu Lys Gly 355 360 365 Gln Met Ile Tyr Ile Val Glu Ser Ser Ala Ile Leu Phe Leu Gly Ser 370 375 380 Pro Cys Val Asp Arg Leu Glu Asp Phe Thr Gly Arg Gly Leu Tyr Leu 385 390 395 400 Ser Asp Ile Pro Ile His Asn Ala Leu Arg Asp Val Val Leu Ile Gly 405 410 415 Glu Gln Ala Arg Ala Gln Asp Gly Leu Lys Lys Arg Leu Gly Lys Leu 420 425 430 Lys Ala Thr Leu Glu His Ala His Gln Ala Leu Glu Glu Glu Lys Lys 435 440 445 Arg Thr Val Asp Leu Leu Cys Ser Ile Phe Pro Ser Glu Val Ala Gln 450 455 460 Gln Leu Trp Gln Gly Gln Ile Val Gln Ala Lys Lys Phe Ser Glu Val 465 470 475 480 Thr Met Leu Phe Ser Asp Ile Val Gly Phe Thr Ala Ile Cys Ser Gln 485 490 495 Cys Ser Pro Leu Gln Val Ile Thr Met Leu Asn Ala Leu Tyr Thr Arg 500 505 510 Phe Asp Gln Gln Cys Gly Glu Leu Asp Val Tyr Lys Val Glu Thr Ile 515 520 525 Gly Asp Ala Tyr Cys Val Ala Gly Gly Leu His Arg Glu Ser Asp Thr 530 535 540 His Ala Val Gln Ile Ala Leu Met Ala Leu Lys Met Met Glu Leu Ser 545 550 555 560 Asn Glu Val Met Ser Pro His Gly Glu Pro Ile Lys Met Arg Ile Gly 565 570 575 Leu His Ser Gly Ser Val Phe Ala Gly Val Val Gly Val Lys Met Pro 580 585 590 Arg Tyr Cys Leu Phe Gly Asn Asn Val Thr Leu Ala Asn Lys Phe Glu 595 600 605 Ser Cys Ser Val Pro Arg Lys Ile Asn Val Ser Pro Thr Thr Tyr Arg 610 615 620 Leu Leu Lys Asp Cys Pro Gly Phe Val Phe Thr Pro Arg Ser Arg Glu 625 630 635 640 Glu Leu Pro Pro Asn Phe Pro Ser Asp Ile Pro Gly Ile Cys His Phe 645 650 655 Leu Asp Ala Tyr His His Gln Gly Pro Asn Ser Lys Pro Trp Phe Gln 660 665 670 Asp Lys Asp Val Glu Asp Gly Asn Ala Asn Phe Leu Gly Lys Ala Ser 675 680 685 Gly Val Asp 690 3 955 DNA Mus musculus 3 ctgctacaag cattgcctag acggacgttc taaaagtgat aagcacccac tgtgttaagt 60 ttgttaaatc tgatagaacg agacttaata gtatctggcc atgcgtgtat atatcatggc 120 tcagtagatt tgttttatgc tccatgtata tgtgtgtgta tatgtatatt ttaatgacta 180 taccataaaa caaagtttat atcatgttgg tgcatggcat tctagaaacc attttgtaca 240 cgagtgaatc taagttttag ggaaaaaagg caatttattt gtagacttct gaagtaagaa 300 ttagtatgct atattaggaa aaggagtgac tattttgaag tatgtcaatt cccttctggg 360 actctattat tgcaaaaatt ggttgctcat tcaaatttta tgccaattac attttatcta 420 acatctacat tgccctaatt tgtactgaag tccttgtata ttgtgtttgg ttgactactg 480 gtagctgtga tgggggctgc attgtttcac actgaagggt tacatttgct ttagcaagtg 540 tttggggtca aactatgtcc aggaacaagg ctgggaatac atagctagga cactgctgtt 600 ggaggcccca ccccagcccc cgaacctgcc cctggcctcg ctccaggctt gagcttcttt 660 attagcttag aaggatgtca acttatccag gatatcatat tcagaatatt catcaaaatc 720 attttaattc tagcatagaa tggactgaat tcctgttggt atatttggcc tatcatcctt 780 taaatgtctc tgataattta ttgatatcta tctttataaa atagaaaaaa agtacttttg 840 tgtaaagata tttgtcttta aatttagtat ttcatatcag cacatcaatg tatgtataaa 900 tgttacatgt taattgtgta aaagattcta caataaatta tttttaccac ttgcc 955 4 7697 DNA Mus musculus modified_base (605)..(6955) N = A, C, T/U OR G 4 ccaaatagag gcaagacctt actcaaaaaa aaaaatattc ttgtgtccac aaaattacct 60 ttgaaatgag tagagtagcc tcaaacctaa aagccaggcc accaaagcca cccaaggaga 120 aagaaagttc cccaccaggt ttcagattca ggaaactaca gtggttctgc accagcttac 180 tagagaaaat gtttttagtt ttaatgtgcc aacttttcaa cttttcttag agtctctttt 240 tttctcttcc ttgtcccctt ccctgctatg tgtgtatgta tgtgtgtgtg tgtgtgtgtg 300 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tctgcctttg gtggtttgga ggatggcgac 360 tgggcgggag cagactcagt tctctagctg agcctgggag aagtgggagg gactcagagc 420 cgcgggtttc tcacacaccg ccttctaggc agccctcctc cagtgcctgc cagccggacc 480 ggaccccaag gcgaagagca gcagtgcaca gcctggggag ccagcggagc aaagacacct 540 ttggcccgat gcccctggcc tcctgtgatc gcatcatggg ggttcgctca gctgtttgct 600 tttcnnnnnn atgcatgaaa tacagtagtt agagcattag tgaagcaata atgaagatca 660 tttgcggggt ggaaaagatg gaaattgata gtttgtgcta aggacagtaa gagaaaaatg 720 tgatttatct ttaattgata ctctgttggc aatttttttg tgtgtgcacc tagctatctg 780 ttgtgtgtaa agtcatctat cctttctctt ctgactttcc actgctcata ggtgctgggt 840 cactcctgtc cttgagtcag tagaagcaga tcttcatcag tccacatcaa caccagctag 900 tcagaaggaa gccactgcca agctccagga acaccatgtt ctgcaggaag ttcaaggatc 960 tcaagatcac aggggagtgt cctttctcct tactggcccc tggtcaggtt cctaaggagc 1020 caacagagga ggtggctgga ggctctgagg gctgccaggc tactctgccc atctgccagk 1080 actttcctga raagaatgca gaagggagtc tcccccaaag aaagacaagc cgcaacagag 1140 tctacctgca caccctggca gagagtattt gcaagctcat cttcccagag gtgagtgttc 1200 tccctttagc aatgatagtg gtatttcaaa attggaaatg ctaggggttt caaaagaaaa 1260 tatttagaaa ttaagtttct cacttttaag agcacggtaa acagaatgtc cttgaaacat 1320 atgaaaactg catttttaaa taacagtatt ctaaattgtg tgcaatcttc aaaagtttca 1380 catcttgaat tccttccaaa agatcattag actctgaaaa attcctgtct ttgcttcgtc 1440 tgtcagcctc tgcttacttt acctgtaaag tggagttgta ttacacctac ctctgtgtgt 1500 tagtatacac tgagtgggga tggagcttgg tatggggtgt ctaatgaagc gaagactgaa 1560 aatgaaactc cannnnnntg tgagcgactg aaccttgcac ttcagagaac cttggcaaag 1620 cataaaatag aagaaaacag nnnnnaagta gcccattgtt accgctatat ttctttagaa 1680 gtactacttt tcctagccta aaatttctca gttttcatgc ccatactctg taaaaggctc 1740 aacagcagtc tctatctctt tcccctgagg ctaattatta acgtgagaac tctgccagac 1800 tatattcccg tgattgcctc cagcctttga aaacactcct caatttgcct tattgagcag 1860 aatttttcat ggacaaatga gaaagacata gtttggtttg aaaccaaagt ctgtgactgt 1920 gaaagtctgg gaatcagctt cgcacccagg tttatctaac tctcagcaaa ttaacagcag 1980 gaaaggagga atgcttgagg ccacgtgtag tatagaaggg gttactctgc attaacacac 2040 acagagttca aggacttcag gctgggagtc actaacaaac aaatgcaaac atgttgagag 2100 aaatattagt tattcaggga tgtggtgtgc ttcctaattc cctgttctca cagttttatt 2160 tattacctgc tgcaattgct tcttctgcga ttattttttc aaggtcttct ttttctgaag 2220 atttcctggg aataattaaa ggtcttaaat taaaagacag attaatgagt gttgaaaatt 2280 tggatcatat acaacatata atgataggtg agaattcttt tccagaagca aatcttgaca 2340 taccatataa gatacaatct gaaagtcagt gtcttcatac atcaactgca tggctttgtc 2400 attcatgtct gcctacatgg cttttaaaac cttatcatta tagtgactga ttctgttgaa 2460 atttgccaag ttattctagc actttaaggg tataaaaggg tatttaattg caaggttcaa 2520 cttgccatta gctgagccaa gtttaaatgc aggtatactt cttctgtttt aaatagcatt 2580 ttaataaaac acagtataat ttgagaattt ttaaaaggag actgttttaa gaaggaaaat 2640 tggccagaaa aaaactttgt tgcatagaaa tatgatgctt ataagtatat aaagtactaa 2700 gtaggaatat tttatccaag tgggctgggg tatcatatta tagctatata ctcaatttgg 2760 atatggagag tcacaatcct tttttggata attcatcaca gtaccagann nnnnnctctt 2820 ttgagctgac ccacttgatg gccgacttgt gctgtgtgct tgcaggtgcc ccagtggagg 2880 cgctcaaaga ctctctgggc gaggagctgt tcaagatctg ctatgaggaa gatgagcaca 2940 ttttgggcgt ggttggcggc accctgaagg actttctaaa tagcttcagc acgctcctca 3000 agcagagcag ccactgccaa gaggcggaga ggcggggacg actggaagat gcctccatct 3060 tatgcctgga caaggaccag gactttctaa atgtttacta cttcttcccg aagagaacca 3120 cagccctgct tctccctggt atcattaaag cggctgctcg catactgtac gaaagccacg 3180 tggaggtgtc cctgatgcct ccctgcttcc gaagtgactg taccgagttt gtgaaccagc 3240 cctatttgct ctactccgtt catgtgaaga gcaccaagcc stccctgtcc ccaggcaagc 3300 cccagtcctc gctagtgatc cccgcttcgc tcttctgcaa agactttccc gttscatttc 3360 atgctggacc gagacctggc catcctgcag ctgggtaacg gcatcagaag gctggtgaac 3420 aagagggact tccaagggaa gcccarcttt gaagagttct ttgaaattct aactcccaaa 3480 atcaaccaga catttagtgg catcatgaca atgttgaaya tgcagtttgt catccgggtg 3540 aggagatggg ataactcggt gaagaaatcg tcaagggtaa ggaaaatgta acgcggattc 3600 aaaataaaac caattgtttc atactgaagg gaaagaaatc acatgacaaa tgagcagacg 3660 ctatttggct aacaaatctg tctaaaattc taaaatgatt taacaagtga atttcttcct 3720 acatccgttt ttgctgccta cttaattgat tgcaagtatt tattgaatac aatttgcctc 3780 tttaaaattg cagtgggtat tgtggacacg cacatctcat aggtagaatg cttgcctgac 3840 ctgcaggtag gcctaagggt ttgatcccca catcctgtaa aactggattg ggttatatca 3900 catatttttt aattctnnnn nnnnacttgt ttctttcatg ctcagagcag ctcatcattt 3960 ctgtgtagca tatcctagta tattcttcat gctcaagagc agctcatcat ttctgtgtag 4020 catatcctag tatattcttc aaatgtctgc atgaatttca gaactgagac caaactgaag 4080 tgattaaaaa gtctattctt ctttagctag ttggaaacaa aatggatgat ctcagcaccc 4140 acttctaatg ccagactcaa aaagattacc ccagtagcca tttcctctct ggtttcggaa 4200 aggattggga aacttgacta atgcatggta acaggttctc ctttcctttg caggttatgg 4260 atctcaaagg tcaaatgatc tacatcgttg aatccagtgc catcttgttc ttagggtcac 4320 catgtgtgga caggctggaa gatttcacag gacgggggct ctatctgtcc gacatcccaa 4380 ttcataacgc cctgagggat gttsycttga taggggcagc aggcacgggc tcaagatggc 4440 ctcaagaaga ggttggggaa gctgaaggca accctggagc atgcccacca agccctggag 4500 gargagaaga agaggacagt ggatctgctg tgctctatct tcccctctga ggttgctcag 4560 cagctgtggc aaggacaaat tgtgcaagcc aagaaattca gcgaggtcac catgcttttc 4620 tcagatatyg tagggttcac tgctatctgc tctcagtgtt cacctctgca ggtcatcacg 4680 atgctcaacg ctctctacac tcgctttgac cagcagtgtg gagagctgga tgtctacaag 4740 gtagggaagg tggaaaaaga acagtttagc aggcctaaac tgtgaccttg gaaaggccca 4800 gcacccgagt gaacaccagt gctgatgaat agctctctgc atttgggtca cacaggctaa 4860 atccggggtc aggcatagtg cattaattat gtgggtgttc tccagatgga aatagctctg 4920 tacaaactaa ttgttcccgg agaggtctgc cacgttgtct tctaaaaatg agcatagaat 4980 gaagcgagat tagggtgaat ggattattct tacagtgaga aggtagannn nnnnnctcat 5040 cttcttggtt gacttggaag cattctatct catttaaact catggtgcac aaaatcagta 5100 tacatggcta tggatttctg ctagtaaacg ttggggagtg tgtgacacag caatatataa 5160 atggtctaac agctgaattt gaaaatgttg tgaaggctta aacacacagc tttctctaag 5220 cttatagaag gccaatgcta aacctgtctt tactatagct ttcttcttaa gttttgttga 5280 ctttcttaaa actagatatt aaaaagttga gtgtatagaa gtctgggtta tttcaatggc 5340 ccattgctga aaagtaataa agtgaaaagt gttctgtaag attaagtagt aaatttacta 5400 taagataaaa ctctggtcaa agcaccagat ttttgacaac cagctaatgt gcaacaagtc 5460 acggccttac cttgataggt tctccgtggg gagacatgac ctcattggag agctccatca 5520 tcttcagggc catcagtgct atctggacag catgggtgtc actctctctg tgcaatccac 5580 ctgccacaca atatgcatcc ccgatggtct ccacctagat acaatataat ttgccccgta 5640 taaataaaag tttagaagaa gacagcataa cagaggcata caagagaaac cgcagtgctc 5700 atgccctaag gagaagcagg gtaagtggag gtgtcagagc aactctttat ttcataaaat 5760 gcataaggtg ccaacttctg actttagggc agtgaactga aagacatcac aatttgcaaa 5820 aactctatca gatcatcaga tggcctctat tgcagattta ctactaatca tgtgttcttt 5880 tcttaaggca aaacaacaac aaaaactata taaaatggtt tttatataga caaatggatt 5940 tgttttttta gtttattttt attcatgatt ttatatttat gtatatgtgt atttgtaact 6000 catgtatgtg tgagtgccta caggtgccag aaaagggcat tcgagttcct tnnnnnnngt 6060 accaaaagga tgtcagttca catctgaata cccactgata gactgagaag gttaacacac 6120 attctgagta aaaagttgtg gaagaaaatg caatgtctgt taagaattca tgccgcaatt 6180 gtctcccaag gacagtgaaa gccttaaagc acattaaagt aggaatgcta aagatgttca 6240 ttccattctc ccagaagatc gccaagagag aaaacaggga tggaggatgg actaagatga 6300 caagtttgtc aagtgagatg catgcatttc aggtccttag tgccatccat acctgtatgt 6360 ggtggggctg acattgattt tccgaggcac actgcaggat tcaaatttgt tagccagagt 6420 gacattgttt ccaaacaggc aataccgggg catcttcact ccgacaactc cagcaaacac 6480 tgatccagaa tgtagtccaa ttcgcatctg aaggcaaaac agcagtgtcc caggtcactg 6540 caaagtcacc tctcacctga cagttttcct tgagtgtcaa tgctagatgt tcctaagcag 6600 taactaataa caccacaccc tttgaaagcc ccagtgagcc acctttgccc atgcaaggcc 6660 acattcggag agcagagtca gcgggcacac ctgctcactc tgctccacca ggtggtcagc 6720 tttaccccac ctgtctttaa catctggaat aaatatgttc tcaggcctct ctggatcact 6780 gcagcagaat aacaaagaat ctataggctg ctcccaggaa cctgtgttgt cctttatatg 6840 aaagaaagat gtccttttat attctagaag gaaattcttg aaacaaagac caagaaggac 6900 atacctgtgc ttccagatat tcagaaaggc taaaagggag taactatnnn nnnnngtttc 6960 tcaaattgca ttaaaatgat aagtgcttgt ttattattaa aaccctaggc aattttgtgt 7020 ttaggaattc tactattaca taaatcacac catccccaat tatcatataa tcaatctgtc 7080 ttttaatctg agtcaaatta tgagtgagta gaattttcca tgaaatttac tcattgtagt 7140 atttctttta tgagactatt gacttatgga aatcctttaa gacctaataa gactagtatt 7200 aatattcatt tatttctgtt gtctttttgt cctgaattgc caagtgtttc tgccagcatt 7260 aagcttccat tttatttaaa ttgttctcag gttactcaaa gactgtcctg gctttgtgtt 7320 caccccgaga tcaagggagg agcttccacc aaacttccct agtgacattc ctgggatctg 7380 tcactttctg gatgcttatc accatcaagg acctaattcc aaaccatggt tccaggataa 7440 agatgtggaa gatggaaacg ccaacttctt aggcaaagcg tcagggrtag attagwgagc 7500 cacatgctct tatgtttgat gcctttgaag gtgtgcagaa cctctgtgtt gaccttagga 7560 ttacagaara waaaaaaaaw cagtgttaaa attacagagg ctaaacacag gtttcctctg 7620 tctcccattt aagatggaaa agaaaagtgt tcacttcagc gcttcaacct tcttctattt 7680 aagaaacaga cctcaaa 7697 5 15093 DNA Mus musculus modified_base (1213)..(8441) N = A, C, G, or T/U 5 acttacttta atgtcttccc caacacmwmr rrwysakrrw tkcsssmwmr cmagcwscym 60 mrrggsmatg ggttccacga aaccsgtamt kaagggmara rarrrarrrm csgtggtcaa 120 gggacccggc gccattgcaa acccgggttc cttccacccr gkgcctytga ccttgtcatt 180 cgccttcaag gataagcttt ygggkaccct attcttaaaa gggggggact ctgcgggatc 240 ttttgaattc tcgtcgtccc tttcccctcc cccatcttct tacccctgct cacctgtcaa 300 ccgtggtgca acaggggtag ctcaggaacc tgagccctga gcttcccaca ccaccctgcc 360 cgtggaaccc agaatcccca gaagttggag gctggggttg agtggtgggg acccaaccag 420 acccagagac tcggagcact ccagcagatc atatgtcccc acctcccacc aaaagcgctg 480 ttcctcgcgg ccctccgctc tgtgtgccct ggggcttgtt tctgcatctc agccagctct 540 ctagccagtc aaggtccacc cgctcaggtg tgatgttccg cttgaccatc ctgacagcac 600 tcaccatggt gtcccgcgcc cggagcctag gaagcagctt ccaaggcaag agtaccgcgg 660 aagggaccca gacagcgact acaagcggcg acagtgctyc cagacccagc acaraagctg 720 aaagctaagg gcatcartct tcggccaact ctgattgcca cctctttcar ccaaygacga 780 gtggagccgc aggaggctgg gcgctgtttc ttcccatatc ttagccaatc agtgacagag 840 gagggaattc agatgggctc agagctactg tgctcaaaca ttttcccgcc ccagtctggt 900 gacaagtcat ttcttttgag gccttaaggc agatagaaca gaaccagctg tgctccttcc 960 agcatggatg gggtattgtc attgtcatcc tcgtacatct gttctaatgt cctagagctt 1020 gggcgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt tgcgcgcgcg 1080 cgcccactgk tkattaacat tgcyactgkt ttttgmaatt mtgcacawaa acatttcctc 1140 tggagcttta cctaacaaca gcgcaaacta ggtactcctc aaaattaact tttttctttt 1200 tgtccattaa gtnnnnnnnn nnnnnnnnnn nnntacggtt tcgtgaacca tgccctggag 1260 ctgctggtga tccgcaatta tggtcccgag gtgtgggaag acatcaagta agtgaacagc 1320 cccgctccca gggccgcgcg ttggcaccga gcccgcgccc gcccgcttcc ctggctgctc 1380 ccagcagggt cgtggtgcgc atcctccggt cycctctccc acgctccgaa gccgggacca 1440 ggagaggagg gtaggccggg gcgtggccgc ktgatcaagc cccttggggt cgctgcggcc 1500 tyttcacccg gactgagcgt gggaacaccc agacaggcag agcactgtga ctcccaggcc 1560 aggttcagag ggttggctag ctcccatgca aggggacctg ggggcttcct gggcctctgc 1620 aaggtcttag gatcagaggc aggaaatgaa cagaagattc gctttctttt gcgatttgca 1680 caccaggaac acgtaatatg gttaaatcat ttggttatgg tatagtacac tcaaagatta 1740 tttctcagta agtcatgaga aacccgatat gtaataaagg gacattttcc ctttaacttg 1800 cccaaatcac ccttgctttg ctgtgttttc ttctggcaac cctagcaggg cattctattt 1860 tgtagagctt ttgagtctgg tactcagata aagacgagaa aatnnnnnnn nnnnnnnnnn 1920 nnnncattag tactcaaaca gctttactcc atgaaaaccc gttttgaatt atattttttt 1980 agccatgggt ctgatctgtg aacttcattt gtcagtggat tgcctctctc tttttgccag 2040 tgcttcacat agcttattac tataacacat ttgaagaata atgtttcctg cacactacat 2100 ttgagtgctt actcttcggg tgatgctgaa gtattggcag tagttttttt gtttgtttgt 2160 ttgtttgttt ttccctccat gcaagacctt gtacagggat agggatgggg gcaggagatt 2220 tccactttcg attttctgtt tacccaacag cttatgctga aaaggtagga tttagtatag 2280 ccacagtcta gcatcatcca tcaacccatc acgactgtac aatgaaaacc tgcataatgg 2340 tacactgtag tcttggagca cacattcctt tatggtccta tgtgtagcta acacagagca 2400 ggaaagttgt tttcaaccta ctgaggactt tgcttgcagc agccaccagg tcatatgttt 2460 tggaatcatc gtatattatt ctgacaagaa actggccttc ttcatccagc tgtgcctctt 2520 ttytgaaaga aaatggaaga gtgatggaga gcaatgttga aataagagca tctgtcaaat 2580 tcatgatggt gtctatttct gaaacaggat ttctggaggc aaaattgctt aagatgacgc 2640 tgtgtaccta acaacccagt gatttcagag gaattgccac atgcttctgc ccaaattaaa 2700 acaatacatg caatacattt tagtactctc atgaataaca cactgcaatg tagctatgtt 2760 taaactggga aatagtaaat atatattgac tccctatagg tgtgaggcac tgtactggga 2820 actggagatg caggctttag ccaaacaaac tggaatcctc ttattttgga acaaatacac 2880 tctaggggga gcggcatgtt cggctaagaa ggcagggagt ggatcaagag agccagcagg 2940 gtgtgtgact gtttgggtag atagcacagc ctactgaaac tggcctgagg gagggtagag 3000 tctctgcatg acaannnnnn naagtaggtt aaaatatttc atatttaaaa aaaaaaaagt 3060 ttttttgcca caaaaggttc ttacatcttt atttttgtgt gtgtgggggg gaagaaggtt 3120 gtgtgttgct cgtgctcgtg cacatgtgtg catgctatac agatatttat gaccaccatc 3180 atgtgtatgg gggcagagga gaacttgaga atcagttctt tcaaccatgt ggaccttggg 3240 catcaaagtc tggccactaa gcttcataga gtcattttac caatgctgtg tccttgattt 3300 gaataaatgt ttttttaaag aaacaataca ataaatttct aataaaaact attaaacata 3360 agtgtgttag tatataaatt gagaaaaatg gttatatccc taatagtgtg atttacacca 3420 ttaactcgtt agtatagtct tttaagattt catagtagtt cgttggtcag agattctatg 3480 ttcacactgt acaataattg tgaatatttt atatcatttc catatacact tgtgctaatt 3540 cttctcattg agagtcttct gcctgttctt tctttttcat ctacaaaaca gacctcaatg 3600 ctggcgaaat cctgcagatg tttgggamsa tgtttttygt cttctgccag sagtctggct 3660 atgataccat cttgcgtgtc ctgggatcta atgtcagaga gtttttgcag gtgagatgtt 3720 cgaggtcctg agagtgaaag ctctcagtgt ctgttattaa atttgagctc tgaaatactt 3780 ctatttagcc agctggacaa gagtgtcttg ctgctggcaa ggaaccctag tgaccattct 3840 cacataaaat ttaattatta gatgattcta cacatatttc agaattttca gtcatgtgtt 3900 ttcatagttt ctcattgttg attctgaata tgcaagcctt caattagtat tctcttaaag 3960 tccacagggt ctatcatgac ctgtccaatt gcgtttgagg aagaattact catgctaatg 4020 gtgtggtctt ttgttttcaa tatgtaatct ggtttatttt ttgtaacata cttctaaata 4080 taagctctca ttggacagtg tatgagtatt taagtaaaca gtttctgggc tccttattag 4140 gagaaagcag caggcgcttc aaggtatcca cttttggaga acttaattca tccgcatttg 4200 gaggttacaa atcagttaat ctaacccaga aaggaaaagg agattttgag ggaaaattcc 4260 aaaaggttga tttccgatta aaaaaaaata accnnnnnnn nngttgtaaa acggcgggcc 4320 aagtgaattg taatacggac tcactatagg gcgaattcga gctcggtacc cggggatcct 4380 ctagagtcga cctgcaggca tgcaagctta caaaaaatta ggaagtttaa ttttctaagc 4440 ctaataccca ttcagatggg tattatagtg cgagcagaaa agaatgtggt tgatttacac 4500 aatgttctct cctgaatact acagtgtgca acaacaaaca atcttagatg cctgcttttc 4560 atcttaagta tgctcttgag acttgccgtt aacaatcaca tagattcgtc ccaactattc 4620 tgctctcttt tcattctctc tgtggtactt acctcagagc catctcaggg gcgccatgaa 4680 ggatccccct aggtaaacgt ttacactagt gatctgccca gcaaacccag atgtgaacct 4740 tctctctgct gtttgcatgc ctatgacctc atagacattc ctagtcacta tgcttccgtt 4800 ttcactacag aacctcgatg ccctgcatga ccacctcgcc accatttacc cagggatgcg 4860 cgcgccttcc ttcaggtgca ccgatgcgga gaaaggcaaa gggctcatcc tgcactacta 4920 ctcggaaaga gaggggcttc aggacatcgt gatcggsmtt atcaagtact gttgctcaac 4980 agatacacgg cactgagata gacatgaagg tagtgttcac ccgctaagat gttccgaata 5040 ttggaacatt tgaaatgata cctcactgat tgctgcttgt gggctacatt agcggtatct 5100 gtgagccaaa atggcgaaaa ccccagaagt cttctctttt tggccttgaa atgcattata 5160 ctgggcgatt tcccccttgc aacagattct ctttctattt atatgttact caaggcaaaa 5220 agaaattcaa ttcctctccg tgaatacacg ccagctggaa ctagggtggg ccatgtgaca 5280 tgcgagttat cagattgtgg agagtgaaat gtaaagaaca ttaaattagc cgattttaca 5340 aatcacttat tccatgtgaa ttctcttctc tttcctaaaa cgtaccaaga acatttttat 5400 catgtcaacc aaaagtcttt ggttctttct tgattttgct ttttaatcaa gttgtgctgg 5460 tgaacttagc caagccagct cagtcttttt ctgatgctat tgataaccac tctttgctat 5520 ttctgagtgg caagttttwa aggtgtcaaa tmcagaagag ccatgccaat ggaaaaaaaa 5580 nnnnnnnnnn nnnnnnnnnn nnactccaag aaaagtcctt tatttatgcc cacatattaa 5640 taatttccag aaattgctgt cttacaaaga tgctcatttt gaaagtacaa aaatatagaa 5700 attagcacat tactcatcct ctgatctaat gtcataagaa gttcagagcc agattaagat 5760 agcacataaa gaaagcaggg tttttttaga aatgtccaat acaatcactc ctgttataaa 5820 gaattaataa tgaggctttg aaaatgcagt ttatacttca gcaaaaataa gcatcgtgga 5880 ccaaaatact gttggattaa gagagggtgt atttggtcca ttcactcccc atgcatgagt 5940 tgaaatatca cgccaatctc agtttatatg tacaaccagc acatattgat caacgatgtt 6000 tactgaagtt ccactggatc tgtttttaac ttatgcggat tacatttaag ttaaaagttg 6060 ttaccttggt gaacagagga agaggatgct atctgcttct ggtaagtaga tcatttggcc 6120 tttgagacgt aagcagctaa tctctgctcc agtcagttca tcctcacact caagtttctc 6180 cacatccagc aacccttcct taaacaggaa agacaaaacg tcagcttctg cctgacaaag 6240 cagctggagg gatcggaaca acttcatttc tcaccagccc taagagaggg aaacgcatcc 6300 cttagaacag gagagggggc attgctccag gtccaaggca ttgttagaag cacagtgcct 6360 gatacccttc tgtgcactca ggaaggcagc agctaagctg ctgtgcctgc ctgcatagta 6420 gcttctgtgt ttttccactc tggccacaac atgcattact gaatggtaca aaactaaggg 6480 aagctagccc tgtattcaca agacatcaaa aagcagcaag tcagcctcaa gcatataaaa 6540 cacatgtaga tgtatctttg aaaaaagtta ttgaagaaat atgaaaatgt gtggatgctc 6600 ggggaaaatt ggtttgctaw aatttcccaa gagcgcaatc gaggggatat ggaaaccatc 6660 ttaactccaa actacatgag gtttatgagc tttcaatcct tttttwtttc ccaagaaaaa 6720 aaaatggtta gccggaatta twaaccaatt tacggaaaaa gtaaaccaca gctgacatta 6780 tcaaagactt cttgttaggg gaaagaagca ttggaacttc aagggtactt gacgttacaa 6840 cgttggtttt attaagtgag actgagatgt tcccagccat acacttgctc caactttttg 6900 ttcactcatt ctcatcagct gacagaggtc cgtgtcccca gtcaaacact gctctcttga 6960 aagttgtatt ttcactttct tttattttta tctcagagac tatgctcaga gtcgatgtcc 7020 cctctctgtt gttgcaggtg gacaaaatgc cttgcttaca tttattcaaa gggttcttgc 7080 ttaccttgct tctcagtaca aagrctgtat tgatgtgtga aagaatccca tggaaactga 7140 tgtcgatgtg agggcggacc agagagaaga cagacagaag gctgcagttc ccaggctgga 7200 gctaagacac agaaaaagag caaagagcat gcagctaggt tgacttacga ttagccattg 7260 gtcttgcaat taagctagac cagttataag tgaaaaattt aatcgtaagt attgtgtggg 7320 agattttccc tgactgtggc ctccaatttt gctatatgca atggagtatt taagcaaatg 7380 ccaaatactt gcaggaacaa ggaaggattc aggcgactac tatgtagata taaatataaa 7440 gctcatgaaa ataaaataag aaagagaaaa aaggagattt atcttctaaa ctgaagatgc 7500 ctgtggtatc aaatgccagg aagtcaaaac accggtaaat acttccttac aaaacatcca 7560 aggagagtca atattaatag aagtgctaac acaaagtgcc cactgatgat gaagcaagta 7620 actgactgaa gggacctcct gnnnnnnnnn nnnngagaaa atgaaagccc caaaagaaag 7680 aaccagcata aaaggcttta ggcagaaaaa aagggatccc aaggaggggt ggctagacct 7740 cgggaaaccc aatcactgtc tacagtagca ttgcactgat aagattctta aaagtcataa 7800 agcaaaacga agtgttttct caatagaagc atcattactc ctgacacaag catggagata 7860 aatttttctt ctggttttta tgataaagaa gaacaaggta agtttgttaa atgcactagc 7920 cttgtattaa tatctaaggc aatttcttgg gagcagcttt ctgcatattc tttcagtgaa 7980 ggtagatgcc tttcaaaagt acaactcaac atgtatttca agtttgctga ttgctagtgg 8040 gggtttagat acactgaaag caaagccatg ttaggtcagc ttctccttgg gtttcctcaa 8100 cctgatcttc tgaatagtga agttggagaa agagaaagtg agatgccact tgctgtggct 8160 gtggctgggg ctgaccacgc aacatggaca ggtggtgctg attttaactc aagctctgtg 8220 tgacagtgtg atgaacctgg acgacctaac aagaagaggc ctgtatctga gtgacatccc 8280 tctccacgat gctacccgag acctggttct tttgggaraa cagttccggg aggagtacaa 8340 actgacacaa gagctggaaa tcctcaccga caggctgcag ctcacactga gagccttgga 8400 ggatgagaag aaaaagacag acacnnnnnn nnnnnnnnnn naaataaatt ttattccctg 8460 tcatgtgagc aatgtttctt ctttattacc actattggtg caaacatttt aagatgctca 8520 tactgacaac atcaagccta ctgctgtcat aagaacaggg ttagaagcta ggactacaaa 8580 tgaacatggc gaagtcctat taatggcctt gtgctttttc ccacctgttc atttgtttgg 8640 ctgattttga gttggtttga aatggttcag gattggggaa tatctgtgac cacggcaggc 8700 aaatggttag tatgtttttt tgttttgcac gtagctaaca attgatgctg gttttaaaga 8760 tgtcttacta aaacccagca caatggcact gatacagtaa agaggcccag ccagagaggt 8820 aaaagctcac cagcatctta cagtactgtc cactaattcc cacagattgc tgtattctgt 8880 ccttcctcca tctgttgcca atgagctgag acacaagcgc ccagtgcctg ccaaaagata 8940 cgacaatgtg accatcctct tcagcggcat tgtgggcttc aatgctttct gtagcaagca 9000 tgcatctgga gaakgggccm tswagattgt cawkctcstc arcgryckcy aswsycgatk 9060 tgayayactg acwgaattca ggcgaaaaaa cccatttgtt tacaaggcaa gtcttcatsg 9120 gagcctgttg tagtaagtgc agtacagttt gtgggcttcc gagactcccc tgaagaagcc 9180 cttggtcact gtattggatt gtagctgact tgctagcatg ttaagagtca ctgcagacca 9240 ctggcttaaa taatttcctc actgcaaaga agtgtatttg ccgggcgtgg tggcacacac 9300 ctttagtccc agcactcggg aggcagaggc agacagattt ctgagtttga ggccagcctg 9360 gtctacaaag tgagttctgg gacagccagg gctatacaga gaaaccctgt ctcaaaaaac 9420 caaaaaaaaa acaaaaaaca aaaaacaaaa aacaaaaaac ccaaaaacca aaacaaaaca 9480 gtgtattgtt tgataatttc atacatgtag ttaatgcact tgatcatatt cataccacac 9540 aagtcttctc ttacctccct gccctctcaa tcttcctgag aagtgcccct cccactttca 9600 tgtcttttgt ttctgttttt tgtttgtttt gttttgtttt gttttgtgat caattcaaat 9660 tgtgccaggg ctgcttgcac aracatgggt aaggagcttg tctactgagg tatagacaac 9720 acaccagtwg ccacaaccac tggaaaaacc atgactcccc attttcctca gaaacaatcc 9780 cacatctata atttaatgtt gacttgccca agccttgggc aggccctggg aaggcaccag 9840 aagctacaat atgatcttga gtataatagc cacatcattc ccagaaggca aaatgtcagg 9900 gaattctcct gcatcatcag cgtttccatt ttcttctgtg ccctctttgc agatgtgcac 9960 tgagatgata cagatgtttc atctagtgct gggcatgcca taatctttcc cagcccctta 10020 gccagccatg agtctcattg ctcactgcag aggggcactc tactgagcaa ggctgagggc 10080 agcacttgtc tatggtataa acttcaacct ggagtagtta tcacttagga aactttcttt 10140 tctttgcagc catgatgagg tttcatggag caatctctgg ctgacacaaa gaatcccgca 10200 gtttctctgc acttaggtta acgagtctgt gcagattgtt tattacattt agtataaaca 10260 cagcattcat tttttttttt tttactcagc caaagacctt tttttttttt ttcctaaaat 10320 acataaatgc tttgcactgg aagaatccat ggagcttgag aagggtcctg ggcctcaggt 10380 ttacttttag ataccatcaa cttgaagata aacatttaca agacatctct gccctttgtt 10440 ttaagtagaa acaaatataa tattggaaaa atgaagtcag wggaatattg ctttataaag 10500 gcaagaatgt tactgagwgt ctctaggctc ctggggtact gdgacttcat aagaaccatt 10560 tatcttgtgg ttcttttctg agccatagkg ctacacttgg tgagcactca gaacaggcca 10620 tggtttagcc aactttgctt attttgaaac aaaaaaagtg ggtggtgcct gaggaatgat 10680 acccaagcct cttttatgtt cacagagatg catacctata tatgaataca caaaataaac 10740 catcacattt aaaagttaaa agtataaata aatgaaaaat taaaatctgg gtagaagcca 10800 ggagtagatc aatgaacttc agtcamcagc tcctatgttc ttggactgct gccacttcag 10860 ggagaaggga agtaatagaa ggttttcctg aggcctgtgc aagcgggagg accctagtct 10920 gcaaaagcta ggatggccat gcacttctgt agccttggtg atgggagaga tggaggcaga 10980 tggatcctga ggagttgctg gccagtcaag ctgaactcag ctctaggttc tgaaagagac 11040 tctgcctcaa aaataaggtc tctcactgkc tctctctctt tgkgtctctc tctgtctctg 11100 tccctctgtc tctgtgtctt tctcwcwcwc atwcwcwcwm agmwwawtgg cawmwmttaa 11160 gggagagaga gaaagagaaa gagagagacm gagacakmgw gagkgwgwgw gwgagwgwgw 11220 gwgagaaagg agagagagag agagagagag agagagagag agagagagag agagagcctc 11280 tatcaacctg tagcctatat tacttaggaa agcaatctat tacccacacg catggaatca 11340 cttgaacatg catgagtaag gacattttta catgtatctg catgcacaca taratattct 11400 actgagttat aagaggattt cagatgaagt gatcaagacc caagctgccg gaaaaaaatg 11460 taaattacaa ataattagaa gattccaacc tgctagagct cacaatgcta aagaggatga 11520 tgatgatggt ggtggtggtg gttatgatga tgatgatgat aaactgtcac tttatgtcaa 11580 ccactgaaca tgtaaatgtg tgtgtcaccc taggtggaaa cagttggtga caagkatatk 11640 wcagtgagtg gcttgccaga accttgtatc caccatgmay ggtccatttg cacacctggc 11700 kkymgwyayk agtggawmyk agctcggtac caagcttsaw gcatagmtkg wgwatctstw 11760 casgygwsta vmtaaatgga agactkggst gctaatagct cagttgataa ctagttcata 11820 ttccttctga gggaagggtt aacatgcaca aagaagagca ttttgaacac aaagagccga 11880 ttgattgcat ttcattctcc catagacatg gtaaatgaac tagctcatct gctctgtgga 11940 cagcatgtcc tgtggatgag gaaagctccc aatatttctg tttttattgt tccatcatgg 12000 aagttaccca ctccttctca ttgccatttt caacttcagg aacattgctc ttcacggaga 12060 gatatttgaa tgaaggcgtt tgagagagtc tataaaacac atatgttaac catcaaggct 12120 attgaagcaa aaactttgct tctgcagata acaatcggga tccataccgg ggaggtggtg 12180 acaggtgtga tttggacagc gggatgccct cggtatttgt cttcttttgg ggaaataccg 12240 tcaacctyac aaagcaggac agaaaccaca ggagaaaagg gaaagataaa tgtttccgaa 12300 tatacataca ggtgaggagg gaaatgtcgc actatttgcc tggtaccttg cgagtggact 12360 gtgtgctttt gctaccttta aattattgct acctccatgt ctcccctctg ctaccacaat 12420 tacttttcac tggagaagag tgtaccactc tttccaccag aagccatctt acctcaccct 12480 gaaggtttcc aataaagcac catcactaaa atcctaactg cttcaaacta caactgcagt 12540 gttggagtca ggctgtctta ttgtctatga aacctttatc aagttccccc tctgtttcat 12600 ctcatgactt tcaggtgtct catgtctcca gaaaactcgg atccactgtt ccatttggag 12660 cacaagaggc ccagtgtcta tgaagggcaa gaaggaacca atgcaagtct ggttcctatc 12720 caggaaaaat acaggcacgg aggtatggct cattakagca gtgctttttt attwttattt 12780 ttatttccct gagagagatg ataaaagtgg gagagttcaa ggcagaaaca ctggaaattg 12840 cacttggaat gaaaagagtt cttgcttgca gaaaggaact cagtaaaatg agacatcact 12900 gtcacactga tgagccagtt agaacaagat ctgctttaga aagtgctgta aggtctccag 12960 cctgggtctt gtgatgaagt agaaagcaaa gcctcaggca gaggacaact ggggtgtcct 13020 tcctcacagt ccaagstcca raatgccagg tccaaatggk ggggtccccc cttgctggca 13080 ccccttatra gatytcaagc aagtctstag gaatggccat attggcctgg cagggtttwt 13140 caatcaggaa gtcaaattat ccatttaagg accgcaraaa ctccagtggg catgtttgaa 13200 gagactcttg gatgacctgg tctcttcttg agttcctccc tgaaaagatg cctagttttg 13260 attttttcag aatcttaaat aactccttaa gggaagatgt aggktcctga gtgctragat 13320 tcccacttta gggataaccc mtgtccaggg cttgttactg tgccaagcca ttgtytggcc 13380 ttttcacaag aggtcaaatt agaagcccaa gttctgttta ctccatcttt gatgcatgca 13440 ctcctcagag ggtgtgagat agcttgaaca catatcattc cctaaaactg ttttcctaga 13500 atataagcaa tattagaggg aatagaacga ccacttttaa atagttaaat tcttttggtt 13560 gttttcactc tgctaaataa cttgcttgtg aaacatgttc atgggtgccc ttcagaaggg 13620 ccaaactgcc tggtgtcatt ttcttagtca taagacccta agaataattt cctctcaacc 13680 tggggtgwag atgttttata gaaactgtat ataaatgcat atcctattgt cagagacagg 13740 gamattgttt gccagacatt gtaggctcat taagactgat ggttaatatt tttckaccca 13800 tttaaggaaa caaatgagga ggatgaaaac tgagtgtcag gttgtggaac aaagagaacg 13860 tcggttgggt tcgggtgaca gtctaatgtg tgtcaagcaa ggagcacttc tttccctgtg 13920 gatagcaatt tctacttcgt gttttagtgg cccaaggctt tctcctagtt acacagatct 13980 cacactatgg tttatttgat tttagctctg ctttcgatta cttttaaggt ctcagtatat 14040 tttccaaagt ttgggttttt gatgtggatg acttgagctg tttcttaaat tctgctacaa 14100 gcattgccta gacggacgtt ctaaaagtga taagcaccca ctgtgttaag tttgttaaat 14160 ctgatagaac gagacttaat agtatctggc catgcgtgta tatatcatgg ctcagtagat 14220 ttgttttatg ctccatgtat atgtgtgtgt atatgtatat tttaatgact ataccataaa 14280 acaaagttta tatcatgttt ggtgcatggc attctagaaa ccattttgta cacgagtgaa 14340 tctaagtttt agggaaaaaa ggcaatttat ttgtagactt ctgaagtaag aattagtatg 14400 ctatattagg aaaaggagtg actatttttg aaagtatgtc aattcccttc tgggactcta 14460 ttattgcaaa aattggttgc tcattcaaat tttatgccaa ttacatttta tctaacatct 14520 acattgccct aatttgtact gaagtccttg tatattgtgt ttggttgact actggtagct 14580 gtgatggggg ctgcattgtt cacactgaag ggttacattt gctttagcaa gtgtttgggg 14640 tcaaaactat gtccaggaac aaggctggga atacatagct aggacactgc tgttggaggc 14700 cccaccccag cccccgaacc tgcccctggc ctcgctccag gcttgagctt ctttattagc 14760 ttagaaggat gtcaacttat ccaggatatc atattcagaa tattcatcaa aatcatttta 14820 attctagcat agaatggact gaattcctgt tggtatattt ggcctatcat cctttaaatg 14880 tctctgataa tttattgata tctatcttta taaaatagaa aaaaagtact tttgtgtaaa 14940 gatatttgtc tttaaattta gtatttcata tcagcacatc aatgtatgta taaatgttac 15000 atgttaattg tgtaaaagat tctacaataa attattttta ccacttgtct acagtgcgca 15060 ttaatttaat aaaatggtat tgacaccaaa aaa 15093 6 177556 DNA Mus musculus modified_base (2293..144567) N = A, C, T/U OR G 6 tcacctggca aaggcggcag ggaaatatct cttactgaca ggtaaaaaaa aaaaaatgtg 60 gatagagggt gccacttctc tttggctgca gtgtgggatg agaagagact tcagagttcc 120 cgcctatctc ccatctttag tttaagggag aataaaccga gttctggact atcagttctt 180 gcaatctggt tacctcccac cctccatttc cataccaatg gtagcttctt cccaggagga 240 gttttgcact aagagcagga gttttgcact aagagcagga gtttggccat gcagttgcta 300 aaagaagctc attactgtca cagctggaaa taatctgtgt gtaatttcct tatatttgat 360 tcctgcttag gaatctcagg gagttccctg cacattgggt gccatttact tttcaacttt 420 gagatcaggt aagagaaaat cagagtacta tgaattagac ttcacctgga aaccagacag 480 tggacctcta aatgtggccc acctgcttga gagtcattgg agggagggag cacttgttaa 540 agtgcagact cccggaaaca acaacttatt tatcaattaa aacagcagag ttgggttcca 600 gagatctgca ttttaaagaa atgccccaag agatatttat cagcattttc aatcccagga 660 gactattgca gtacttccct aagggttggg gtaactctat tagtatttgc ccaccggatg 720 gcaagctcta ctaatagcta acatttccat aactttgtgg attgttagat aagcatgtga 780 attcataatt tccttacttc ctggaccatg gacatattta gggaggcaga gaaacaccaa 840 gcttcctaga accccttcta ttttgtttgt tcctgtctcc taggctcaat aggttagagc 900 gacgagtatg catgattcag gacactgagg taagaacaga agagatagat atcccctaaa 960 atgccagacc atacaccaca gggttacatc tgggaccaag gagaaaggat ctttattaag 1020 aatctgtgcc aggaaatagt agctaagcca gaagatgaat accagaagat gaatgataga 1080 attgggatct agaaaagtca atcaattggc tcatagtggt tttactgcac tgatactcaa 1140 gtgtaagcac tatgttcagt gtaatttatg ataagtaaca gtattgattt tcattcatgg 1200 gttacaaatg aggtgtgact catcttctcc ggattgtttt tccctgcccc agagatgaac 1260 actaccatgt gccccttcac ttctctttcc cagccttacg gtttcacgga tggctctttc 1320 aatgctcatt ctcacctccc aggatgaact gcttccgtct acacattcct gatgttcaaa 1380 tgtaacgaaa acatggaaag gttaattcat gttgggaaac agatcaagga ctttgatggt 1440 agtagaagga ggttctctac ctgtgagaag gaaggtagga aaaaaacttt agaccctata 1500 atgcacctac cagcacaaaa agcaaagata cttttttaac tccccaaatc aaaaacgatt 1560 aaataatgct atctccttgt aaaaagtcac cccaaatcgt gatacttatg taatatagga 1620 agtacaacta acccaaatca atgacatttt gcattccctt ttatggagga aatgagtaga 1680 atttacaaat agtttatgtt tctctcacca tctcttcagt catctttatg atattgtgag 1740 ctatccaata tttatgtttc ttttcccttt aagtcagcta tagctggttt ctgttgctgg 1800 tgatgcacaa ctctggctaa tggagaaacc aataatttcc ctaatgatta atcctcggga 1860 aatgtacctt cttttaaaga aagctgtggc tatctgtgag aggggaatcc taccttatat 1920 ataagcagca ccagtattgc cattttctca gtgaaacaca gcatggccaa cagtagaata 1980 tgagattgac acacacagta ctaggatata tgtagaagta gagtgggaga ctttccacta 2040 ttattgatat ttctgaaaga ttaacataga aatgtaactt ctaggtctgc tttgtcagct 2100 cagactgcta taacaaaata ccatagactg ggtggcttaa acaacacaaa tcaatttctt 2160 acagttctgg aaactgagaa gtccaagatc aagatacagg cagatccagt tcttggtaaa 2220 gtcccgcttc ctgacttgca gacggttatc tttttgatgt atcctcacat ggtagagagg 2280 aagctctggt ctnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2340 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnatgactta 2400 cagtgtctcc acatgatagt tcccccatgt gacaaaaaac atcaaaatat caacctaaaa 2460 tatggaaaca aaatacttaa actggtttgg ccacatttaa ggaaccatta atgatacttc 2520 acttgagaag catcaatacc ctattgaaaa aattggaaag taaagcaata tttgttccag 2580 tggaatgatt ctaatgcttt gtagttaaaa caaaaacaaa aaacaggaga ccttacctct 2640 aaataacctt tgtggtttaa aagtactttc acaggggtta ttccttatgg ttttctatca 2700 ggtagatagt atgttgctgt gtttgcacat gaggacctgc agcccagagc agtgaggggc 2760 tcacacatta atcagggtct tcacttgcct cattgaccca ttgcagatca cctaacccaa 2820 acccacaact ctttcctgaa gatatcattt tttttttcct ttacgtgtca cgagaatcaa 2880 cagttggtgt tttttgttat aacacctagt agagtggaaa gaaattagaa gataaatatc 2940 tgcaacatta tagatatgta aattgggaga attcacatca tttccctaaa ctttactttt 3000 cttgtctgta aaaataaggt aatacaaata gcaacttata tagggttttg tagggttttt 3060 ggtaaggatc aaaagagtta acacttacga aagcatttca tgtatagtaa tatataatgt 3120 acatttaatt ttctgtcttt attatcaggt tctctatttg aattatatga attttaaaca 3180 ttcttaaaat aagaaaatac cacaaaggag ataagagcat gagtggtact cataatggta 3240 ttctagcttt tggccctgct gtggacaaat tgctgaacgt ctctaaacct tagttttctc 3300 tcctgaaaat aaagcaatta cagagattaa atgtgatgat atctataaag gacatattac 3360 agtgattgat acatggtgat atggtttggc tgtgtctcca cacaaatctc gtcttgaatt 3420 gcagctccca taattcccac ctgttgtggg agggaccctg tgggagataa ttgaatcacg 3480 ggggcagttt cccccatact gttctcatgg tagtgagtaa gtctcaccag atctgatggt 3540 tttataaggg gaaacccctt tggcttggtt cttattcttt cttttgtctg ctgccatgtc 3600 agatgtgcct tttgcctttt gccataattg tgaggcctcc tcagccgtgt ggcactgtga 3660 gtccattaag ccttctttct ctttataaat tacccagtct tgagtatgtc tttatcagca 3720 gtgtaaaaac ggatgaacat acaccgtaag tgtgactttt atcattcatt taagcatcct 3780 gtttataaaa atgtaaataa tccattttca catatgctgt gttttcgata aaataacttg 3840 cttaaatcca atcctggcat catagataat ttaagccatg gttttctgat cccgtcctct 3900 tttgtgactc cagtcccaca gacaggaacc aaagccagca gaacaaagca gccccagatg 3960 tgttctactc tattatctga agtaaatgaa aatcaaccct gaggtatgtt tttatattct 4020 gtctaccaaa ataatgtgca ttctgtttat tgaactgctc ttatttcttc cttgcaatct 4080 gttactttca ttttcattcc tgccaagaga gaaataaaaa agttgctcat aatttctaag 4140 gcgatttaga cctacttttt tggcctgtaa tggaaatctg cggtttcttt agtctgatcc 4200 taggtaaagc tgattttagt gaagccaggg gtttttacac attaaaaaga aaaaaatgga 4260 atggtggaat aaactgagtt agggagtcct gaggtttatg tggattctct tacctatgta 4320 accgctaggc atgccatggt ggatagaata attcccaccc ccacacgtct tccctgacaa 4380 gatgtccata tcctaatcct cagagcctgt gactatgtca ccttataaag caaaaggtac 4440 tttgcatatg tgattaagtc aaggacattg atatgcaaag attatcctga attctgagtg 4500 ggcccaattt aatcacaaca gttcttataa gggagagtca ggagtgacaa agtaagagaa 4560 gcaggtctga tgatggaagc cgaggtcaca atgacgccaa gaagaggcca tgagccaagg 4620 aaggcagagg gcctctggat gctgcaaaag gaaaggaaat agacctcttg gaacctctag 4680 aaggaaagca gccatgctga cacttagata tttggaattc tgatctctag atctataagc 4740 taaaaaaatt ctgttttttt ttaagtcact aagattataa caatttgtca cagcagcagt 4800 aggatactaa ttcagtatgt caagtagctt tcctaaccca aattctgaga catctcaggg 4860 acctgggtat ggactaaatg tttgcgtctt tctaaaattc tatgttggaa gctctaacct 4920 cccctccaag gtttttaggg cctttgggnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5040 nnnnnnnnat cttgactaca tattgtcaaa ttgctccctg acataactgt accaatttat 5100 acaatcaata gtagcacata aaaattcaga aatcccccat tatcttgtga taaagatttt 5160 tccaaactaa tttgtgagca ttgatttttt gctgtaagag ctcagtaggc attgaagggc 5220 agatggaatt tgagatgtcc tagagaacag gaaatcatgg aaaagtagga agaagctgca 5280 gctgaaagat gactgggtat ctgtgtaatt ttgattggat tcagaagata gcaaacctgc 5340 tacagaaatt gtgatgccaa aagagtcaac agggtgaatg aacagtcaca cttatatttt 5400 atcacctgta acatgatgga gggtatattg tatgactttc aggtaggtat tggccaatct 5460 acctaatata gatattgacc agcttctcag gttgttttgt tgtggtgtga aaagataatc 5520 gcagtaaatg tcatagttac tagggaaaga gtattctaaa taagaataga tttcagataa 5580 gtgttggtga atgaattgta ttttaactat ttttctataa atagcgatcc tcagaggttt 5640 atgcaatttt ttttatcgtg aacaaagaac aaatatttga aatcattcag aaaacaaata 5700 aatgtatgta gtcagcaaga aataaatggc cagctttaga aaagatgcct aatgtaagga 5760 ctgttgtatt tactttgttg cttatcttac tgagtcattc cattactaac attctgaaag 5820 cctagtagag cttcactgcc ttcatctgaa ggcagagtga gtgttggcat gtaagacatt 5880 acctttagat ttaaggtgat aggaagtttc cttcgaagtc tcaagactta gcaagaattt 5940 aaagggtctc aatggaatgc ctatatccaa aatcagagtt aaggatgaga aatgggagga 6000 agagaagtca gatgagaagg gatttgtatc tcaaattttc actagaatca actgagataa 6060 attttaccag agttggaagt aggagtaggg aacaaataag aaaaaacatt aaacactacc 6120 aaagtgccca acaatttcca taactggtcc agtaattatt cctataatag tgtccggcaa 6180 cttttatgcc aatccattat ttcatcagat cgtacacatg tgcactaatt aaatgcctgc 6240 agaaagaaga ctgtaagtta attttaaaaa ataactcgaa ggattcaaat gtcaatttta 6300 aatgacattt agaaattcat aggctaacaa accacagcat acaattcagt tatacaggtt 6360 tctgtcagtt accactaaga tgtcttgaat aaattaatat atttttctaa atcactggca 6420 gagtttctgc cattattaat gttgtagaaa aaaaccgagt ccttgtcaca tgaccatgaa 6480 aagttagaca cacagacact ttgaaggatg aggggggaac aaaatttatt ggggggaaag 6540 gagaaaacac tctcagcaaa gcgtgaaggg ttcccattaa taggccccca tctcacagat 6600 tgaatcccag tcactaccca ggaataagag aagacagtct cctcccctct gcaaagggcg 6660 ctcaaacgtc ctgaggctcc actctgtcct cctagtgctt aggtgggcat taatcagaaa 6720 gaatcagttg gaaaaggacg ggcctcatgc ccgaccctgc agtctggttt ttcagccttc 6780 aggctgtttt aggcttgaag gcagggtttc tctgggggat tcttggctac ctgctctctc 6840 tgtcattaag gacaagtcaa agtcaccttc aggttatgtc ccttgtcctg ctatgccctt 6900 tactcaaagg gtaattgctt gcaacagaaa tgtactatat ccttgtttct ttctcttgaa 6960 ttctagttgg gaaacaagcc ctcacattaa tttttttttt tacaaagaag aaagaaaaga 7020 aaaagaaaaa caatacatga tttattttca gactgtttat tgtaaaaatt agttcagaga 7080 agataaagag aaataattac taaagttttt gagtttctca aaacagttga atcctgcaga 7140 ctggattgtt agaacttggg taaataaaga gaaggaagct tgaaagcaaa tgccagcagc 7200 agcaaatttg catttacata gtgtgtttgc aaaacagcaa gaagattagc ttagaagaat 7260 tggaagggga ataaaattga gtaatcagaa gtccttttgg atcaaaagag aatgaataga 7320 ttatggggat ttttgaattc ttatccaaga tcttcaccct accacaaatt aagaaagagc 7380 tactgcagtt tcttaaatga ggtggtcaga ttgcagttac ggccagaaag gaaagtaggc 7440 agtatagtgc caaatgttgg caaggaatat aggaatatga gaattctcat gcactagtga 7500 tggtaatgac cacaggtaca gcaattctgg agatcactgc gacagtactt aaggcaaatt 7560 aaatatacac ataattagga cccagcaatt ttactctcgg agaatgtgtg catgtgggta 7620 tgagtgtgtg tgagtgtgtg taccacgtat gtatgtatat aaaatttctc tagcaggaat 7680 tctcacataa aaacatgaag agacacatac atcacaacat tgggagttgg gcacattctg 7740 ggatccatta gtaaaaaata ttactttgta gattgttata ttgatgaccc ccatacctcc 7800 cagtattcat acgctttggt aatcttcttc cacattgggc ttggcattgt gtttcatttt 7860 gaccaaatgg gtcaaatgtt atgtagaggc ttattaagag cttgcattta ggggcttgtc 7920 cctttgaact acttggatcc agctgccatg tttaggtcac atactgttga aaaggtcctg 7980 tggaggaata ttgagatacg taaaaaacgg ctaaagtctt cttggacagt tgccagctaa 8040 atatagctga atgagtgact ctagccaagg ctatacagaa cagaagcagc acccagatga 8100 atatgctatt ttaaaccaca aaatttatat taaaataaca tagagatatg aaaatataca 8160 gcaaacaagg gaaggataac aacaacaaca aacaagagaa agagagagag ataaagagag 8220 agaaagagaa gaaggaagga agaaaaccaa gtggctgctg ctacattgga aattgcatga 8280 ttgttgtctt gtgaatgatt ctcttaaagg tgtctataag cactcattat taacatgatt 8340 tagagaaggg aacgacacnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8400 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnta 8460 acaatattaa gccaaggaag aaaatttttt gtgagaaata ttgttttgca taaaacaaaa 8520 gtgttttgca ctgagtttat tttcaatttt attttatttt ttgagacaga gtctcaattt 8580 gttgcccagg ttggagtgca gaggcacaaa catggttcat tgcagccttg accttctggg 8640 ctcaagcaat cctcccactt cagcccccca catagctgga actacaggtg tacaccacca 8700 tgcccagcta atttttgtat tttttgtaga gacagagttt taccatgttg cctggcctgg 8760 tctcaaactc ctgagctcaa gcaatctgcc taccttggcc tcccaaagtg ctgggattat 8820 aggcactagc cactgtgtct ggcttatttt taattttaaa ttgaacttca aaactcttct 8880 aagttttcct ttcaaaaacc ttgcttagct agaatttgac ttactctaat ttaacacaac 8940 tggtaagagc ctagtagaag aaaataacac caaaaatata ttaatttgtt taaacaattt 9000 aacatctgct aaccagttat tagatatgtc tttcttatgt atatcaatgg ttctaaatgt 9060 acttctccaa gttatatgac ccaaatgagt aagtttcatc taaaatccaa gttgatgcct 9120 gactcaggcc ttatttgagt cctgccaaaa cagatccatt tctcgtacat tttacagatg 9180 attttatgaa gaaggaaaat gaggaataat gtctgccttt tgtattcttc tttgagcaaa 9240 ttttaatgtc ttaccattct ttaattaact aatgaaacct ttgttgtgtc tttatatttg 9300 tatgagaagc aatgatttta cctttggtta aaaatgcttg agcaattttt tcttgcatca 9360 aacaaatttg aaataaacaa agaatcacag tgattataaa catgaagacc caatatacga 9420 gttactcata aacaatggat caaatagcat cagtagtcac tacatccaat tattatttct 9480 gattttagtg gaagaaaata aaattctgaa gtttttacca aatgaatata aatttttgat 9540 gaattttgtt ataaagatat tttagtgaaa attttagaca caaagacatt tgaaaagcac 9600 tagaattgat accaaagaac caagtcacaa acagtattac aatttctgga attttttgtg 9660 aaatgtgact ttttatgaat ttctgccaat tgtattctta ggagtttgtc tttctgtttg 9720 tataaccatt gcttcatgtg aaagaaactt ttgaaaatta aaattaataa gtattcttta 9780 atcaattaca agtaaaacta gactaatatg gctgtgctat ggaacatcaa tacatggata 9840 aagttaattt tgatgaagtc gttgacaaat attcataaag aggctaaaaa atggaaatac 9900 aatgctatta ttcatttctg tgtaaaacta aagaaaatgt ctgaggggag ccccaatcat 9960 tttagaggtt tattttgcta aggttgagga tgaacctggg gaaaagaaac aaaatcacag 10020 aaatatctgt gatccatgct ttttcctaag agggtttgga gacacaatat ttaaagcgga 10080 aagcgtgggc agtaggggaa aaaggaaaga acaaaaaggg gagggtggat aataaaggca 10140 agcagttgca ttcttttgaa gctttgatca atttcactga atttacattt tacctgtgaa 10200 aggagtgggt agtagaatag tcaactatgt gtttgtctgg tgctcagtga atcttcattt 10260 ttatatagga taaagtaaac atagaccaga ggaagaagtc aaatacgcat ttgcctcagg 10320 tgagtagagg gatgacttct agttctatat ttgtcctgta cctgtgaaga taagctgttg 10380 atttatattc tcagggtgaa attcagtaca acttcatttt acagtaagga tcttggggcc 10440 cgcaggagat tttctgtgag aaaattgtaa gagagggccc ctgagaaggt atgtgccttc 10500 tatctttgca gttatctatt taggaacaaa atgggaagca gttgtgtgtg acgcagttcc 10560 caagcttaac ttttcccttt ggcatcgtga gttgggggtc ctgagatttt attttacttt 10620 cacaactgga acagatttta aaaactttaa aaaacaaaaa tatgtcagtg tccaaaaagt 10680 attaatgcat tactttttct tatttttgta ttgtttattt tattttttaa catttttatt 10740 gacatgaata tataggccat tttcattatt tgtttgattt catgactatt accaaaaaaa 10800 ttttgtcata tagaggaggg aagtattaaa aagggatctc tgacatacac ttggtatagg 10860 gtcctggtac caattgtgaa aagtaaaccc tttcttataa tccaaagtca cttggcaaag 10920 tttcttcatc tcaggtgtcc attttctact cacaaggtgg ttaaacaaaa aaagtataga 10980 aattgggatc tatcagcaga cctgtccagt ttctaacttg gtaaactcta attaaattac 11040 agtgttaagc tttacttttt tttcaaaaga attagagaaa atgcaactta gaacaatggg 11100 cttcaaacta gaatgtgcct atgtctgagt gttctcctgc acagagaatt ttaaggaaat 11160 aaatgtttcc atcctctttt tacaatggtt cttctcccat atatagaaaa gtattaactt 11220 gtcacccatc acaaatctta acacagaatg catactggtg tgtaacaacc tcagggctcc 11280 taacatagga atacaaattg tattttgctt caggggaaga ctcctttaaa aattaatcaa 11340 agctcctaaa atcttgtctt ggcttgtcat acacatattt gtagctagag tgaagttctg 11400 cgttagacac agggtatttg gcccttgctt aagactgaat tgacaaagtc ttaagtagaa 11460 taatgaaagt tgttatttta aatgtaacta gactatttgt taaggcttcc acattttggg 11520 ttatttgaat ataatccaca gataaaaatt taaatgattt ctactggtgt attttagtca 11580 ataaagcagt taaaatttca tgaaaatata tgaaatgttt attcctatta cagtaaatcc 11640 ttatcctatt ttatctcata aatataatga tatataatac ttatgtttgt gataatataa 11700 tttgatataa aatacaatat ttttcacctc ctaaaaataa taaataaata tacttagggt 11760 tatggctaaa aatgtagaca tatgcaagtt aaatacacag tttttcaaat tttttatata 11820 gcacattaac taaaatattt gaagatcatt ggctctgaga actagaaaat agcttgagta 11880 cttttcctga tctgtcatta atttgctgag caatgctgag tataatgttt aaagtgggaa 11940 tccaaattat ctttctgtaa aattatactt tgggagctgg agataccttt catgcctgta 12000 aactgatatt gtcttaattt tttgttccct aaggaagaat aattttctta taattaagaa 12060 ttttcctgtt ctatcattat agcctctaaa atattaaatc ttcttttgtt ggtctggata 12120 cttatatcta actgaactag gatatattat ctttttttca atgtcatcta acacaaaagc 12180 tgtaatcatt gatttgagat ttgagctatg caaaatggat taggtttcca tacccaaaat 12240 tataaaagta cttagatcat aggctattaa gtatttttag acttcttttt ctttttaatt 12300 tagctgttta cagttacaac tttccctata agcactgttt tagctgcatc tcaaaagttt 12360 ggtatgttgt gtcttcattt tcattcatct caaagtattt tctatttttt ttgtgatttc 12420 ttctttgact tctttgttat ttaggtgtgc attgtttaat atttacatat ttgtgaatat 12480 cccacattaa ttttgattat tgacctctaa ttttattcta ttgtgattga tnnnnnnnnn 12540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ngttatttag aggtaaggtc tcctgttttt 12660 tgtttttgtt ttaactacaa agcattagaa tcattccact ggaacaaata ttgctttact 12720 ttccaatttt ttcaataggg tattgatgct tctcaagtga agtatcatta atggttcctt 12780 aaatgtggcc aaaccagttt aagtattttt gtttccatat tttaggttga tattttgatg 12840 ttttttgtca catgggggaa ctatcatgtg gagacactgt aagtcatagt taaagatcct 12900 tcaaaatata ttctaaatgg cattttatta cagatgtatg ttttataagc ataccagtaa 12960 atacactcaa tgcaattctt ttgcttcaaa tgtcaataaa acttagtaag attttaatac 13020 attagcataa gctaatttta actttttatg ttgctgatga agcacccatt caggaaaaaa 13080 aaagctcatg ctttatgtaa acatattaat ggacaattta tctccttttc attattttta 13140 ctcatgctat atttgttaga aaactgataa tatagttgta tgttatgaat ctagttagat 13200 ttggatttta ttttcaacaa caacaacaac aacaaaacca gtaatctgaa catgaaaagt 13260 cgatgtaggc aagagatttt cttgcagatt gtcagttgtc actcaatgtt tcatatgtga 13320 gtccttggct tattgttact aactccaaac aatcggtaac agagttatgt agtcctttaa 13380 attttacaag aagctttcaa agatgtctca tttgatcctc ttagtatctt agttgagcaa 13440 ggtgggccag gcaatgctat caccattata aaatgggaag ttccaggctc agacaggttg 13500 aattgccaag caagtaactg acaagggtga tctagatctt ttagtttgac atggtgcagc 13560 tcttttcact atgcattcac tgtcagtgga catattacat ttatataagg tggacaaaag 13620 gggtttagtt tcattaatca ttgacttcaa acacagcaat aacaaccacc acctcaaaaa 13680 caatattaac taacagtttg tgaggactta acattttgca atcattgctt taagcatcac 13740 acttgcaaac ctgtggggta ggtaatattt ttaaccttct cagcactgga gtatattgat 13800 ctgttatttt tcctgtttta caagtgagga aactgtagtt caaatacatt aagtaatttg 13860 tccaatggtt gaaaacaggc agtctgacta aagaatctgt acaattaact cctacactaa 13920 actgtcccca tagctaattt ttgtttttta aatcaaataa agttacaaaa agaagcaacc 13980 aaatatttta attagtggct cttattccct gctattttct atttatatct ataataataa 14040 gtagtttcaa catagactag agaagaacat tattttaata tagtatctag aggcttttga 14100 gagttatgga cagcttttga aatctagatt ccaacttcca ctgggcgaga ctatgggaaa 14160 gaaagtatat ttagctctac tttgatggga atcttaatat cctccacact ttctgaggac 14220 aacaattcat aactttacta attatacttc ttggctaaga ttttaagttt tcactgttcc 14280 ccaacaagtt cataaagaga tttccagata taggaaaata tcattgacta aatgacctcc 14340 aaaatacctt tcagtgctca aagtcaatca atctttgaca taatcatgtt agatctatcc 14400 ttactaaact aaaaaaaaag attaagataa tctctcccag ccatgtacaa gcttaggctg 14460 taaagaagat gaaaacaagt tagtttttat tttaatcaat ttgacttcag aagcaaaact 14520 ttatagctca atctggtatt cataagcttt tcttgaaaat gaaacatatt tttatttaat 14580 ttcatatact cccagtatgt tatagtgatg tgttgattta acttaatgct tttacttatt 14640 ataacattga tactaagcat catctttaat tctcttttaa tttcttttaa ttttcataca 14700 taattgttta aaccttataa ataaatggat atacatatat atgtatgcat gactaatgtt 14760 ttttgtttat aagttctgat ggcaaggtaa atattaaaag aatttgatgt agaaaaaagt 14820 aatttaaaaa gtgcttcttt taatattcaa tactaccttt ttcatatgac aaaacttaca 14880 gttcattggc aagttaacaa taaatctgaa tgtcacttct ttaggaatct gaattcttgg 14940 atctctcttt gttctattga tctagaatca catcaccgaa tgtgtcattt agaatttgct 15000 tttctctttg cttgtaagta ttctgaattc cttgcttgaa gtattgagtg actatattag 15060 caaaaataat gacaacagat gtgattattg tgcgactaac caagaccagc agcagaagct 15120 tcacaaagaa tcaatattcg gctcttagcc acagagatta aagaggcaca tgaatataaa 15180 gcaaatcatt gtataagaaa tagttgaaat gaaatgacat gctgctccat aaaatgatct 15240 aaaaatggaa gccacagaaa aaactggcaa aacaccagag ctttgacaaa cacatgcaca 15300 gacctgggaa cacccccaaa tatatatgta tcttccttta tccttaatca aaggtttcta 15360 attttcacat taactagcaa agccttgaaa acaatgatta caacacttta tttatttatt 15420 tatttttgag acggagtctc gctctgtcac tcaggctgga gtgcagtggc gcaatctggg 15480 ctcactgcaa gctccgcctc ccaggttcac gccattcttc tgcctcagcc tcccaagtag 15540 ctgggactac aggagaccgc caccacgccc agccaatttt ttgttatttt tagtagagac 15600 ggggtttcac cgtgttagcc tggatggtct cgatctcctg acctcgtgat ccacccgcct 15660 cggcctccca aagtgctggg attacaggcg tgagccaccg cgcccggccg attacaatac 15720 ttttaatact taacttacca aagtaattta tttttattcg ctgaaaatgt gagtcacttc 15780 agctgacatt tctgaaagcc acatattgcc tttttactta ttgttcattc attagactga 15840 acttcatgtt gattttaggg ctggctaagc cattccctaa gctcttcttt ttcatcttta 15900 actgatcctc ctcagcttgg ggcatacata caagaatgca atgcattgtt cagagaatat 15960 agcataatct acttcctgat atgattccta tgctacttgg ccagcagtga tgacaacagg 16020 tagggagctt ttatagaaga atggtgaccc ctcttttgag atcctctaca gctggcaggc 16080 atccttctgt ccctgaacat cacagcctgt gtaatatctt gtaggccaaa caaaagtcag 16140 taaatgttta gtgtctagaa cttcttttag attcagaatg aatataagac aattggaaaa 16200 taaaacagta aaaacattca caaatgcaaa aatgtgttgg acaaaaacaa caacaagtgc 16260 aaaaagttat tggacaaaag ggaaagtgag ggtagaagaa ctgaaataga gagaaagaga 16320 cagagagaaa gggacattgg cagaaatgct cattattcat ttcctctcat atctttctct 16380 aagtgagctt tcctcatttt tcattcccag agaacaattc aatctcctta tcttttgcct 16440 atgtaaagtt taaggtggag gacaccacaa cacaccgtaa accagagagc atgggtccat 16500 tgctgctcaa aggtgaacag ataagagaca tagctttttt ttttaagttt ttttagcaag 16560 ttaaaagtta ttttatgtat attattaaaa agtcaaataa caacagattc tggcaaggtt 16620 atagagaaaa gggaactctt atatagtgct ggtgagaatg taaattagtt cagccattgt 16680 ggaaagcagt ttggtgattt cgcaaagaac ttaaaacaga aacatcattt gacccagcaa 16740 tctcattatt gagtatatac ccaaaggagt ataaatcatt ctaccataaa ggcacatgca 16800 tgcatatacg ttcatcacag cactagtcac atggaatgaa ataaactatt cacaaaatga 16860 atacttgtga tatatgtgaa ttaatatgtg aattcactat atgcaaagac atggactcaa 16920 tctagatgtc catcagtggt agactggata gagaatgtgg tacatatata ccatggaata 16980 ctatgcagac atcaaaaaga atgagatcat gtcctttgca tcaacatgga cggagctgga 17040 gactattatc ctatgtaaat taacagaaac agaaaaccaa ataccacatg ttctcacttg 17100 caagtgggag ctaaacacgg agtacacatg aacacaaaga agggaacaat gaccactggg 17160 gcctacttga ggatggaagg tgggaagagg gtgaggatca aaaaactatt tgttgggtac 17220 catgcttagt gcttgggtga caaagtaatc tgtacaccaa acccccaaga catgtaattg 17280 gcctatataa caaacctgca tatgtacccc tgaaactaaa ataaaagtta aaaaaaattc 17340 agttatgtaa cagtaaagaa aattcatctg tgtagaaaaa taatcaaaag atacagaaag 17400 ttataaaaag taagagtcat ggccattcca gccactggtt ctaattacag acgatagcca 17460 tatcaacact tcctgtgttt cctcccaaac cattattctt acagcaacat gtatgtgcct 17520 gtgttattac ccacatagca ccttactgta tactaactct aaaaaatgga aaatgattac 17580 attcttgtgg tgttttgtaa acctgacgat acttttctgc atcatcattt tctagtcacc 17640 ctcctgcttt ttcatggttg tttaatgtgc cattttaatg gttcaaaatc agtggttaat 17700 tgttgttaaa taatgattaa tgttttaata gttgtttaaa acaaggtgtt agattgtatg 17760 aaaggctgta atctatttac tcattccacc aataatggaa atttttaaag taatttgttg 17820 ttttgtttgt ttgctacatg ccactatttc tgtatcagtt tnnnnnnnnn nnnnnnnnnn 17880 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17940 nnnnnnnnnn nnnnnnnnnn nactttgaca atagaaaatc cctatactat gggaaacatt 18000 ttttgaaagc ccaacttata gatgggttct tctgtcctct caatgattct gtaagccatt 18060 taatacccta taataaattt cttccaatgt aaacaagtta gagaagattg tgttctctat 18120 atagattttg ctttctctct ctctctcaac taacccccaa ctgatacacc atggttttgg 18180 taatcaacta tacgcatttg ctcttgtgac atcttttgga caatatatca tctgccttac 18240 cttgataggt tctccatggg gagacataac ttcatcagag agctccatca tcttcagggc 18300 catcagcgct atctgaacag catgagtatc actctctttg tgtaatcccc cagctacaca 18360 ataggcatcg ccaatggtct ccacctagac ataatatatt ttccatacta taaataaatg 18420 ctcaggaaga atcacagcct acatcggata gaagaaatta agtcatatag cagaattctc 18480 attgctcagg ccttctggag gatcagggtt aatcagatac gcccagaaat ggctttcttt 18540 ttcaggaaat gcgcaaggtt tagacccatt acattgaggc actgcactga gaggcagtac 18600 agtcctatga acacagtaac agttgacttc tactcccaga tttgcacact gactcatcat 18660 atcatttcat ctcttttttt tcaccatata agatagtttt tgtagaaaca atatattttt 18720 gaattataat aaattgttta aagatgttgg gatcatttac tcgttttata tcctcctcat 18780 cataaaatag tattttgcct caagtcacta ctcaataaat gtcacatcaa tgcagtaaaa 18840 ttgtgcaatg taaaaccaag atattaatag tcataagagt aaagagaaac ggttcttcta 18900 aaggaccctt ggggtcctca tagcaattac atatctaatg gatactaatc acaattataa 18960 aaaccaagat gttgaaccag caggtaaaaa caacggtggt gtttgaaaga ccactggaaa 19020 tcaagctatt atcaaaaatc taagtattaa ccttatcatt atcatcatca ttattgttgt 19080 cgttacaggc aatttttatc tgtctgtgat gctggctgca aaaagcagat agttaatttt 19140 agtccagaaa taacccccta attctaagag cttcattttt cttcttctcc aaatgtttat 19200 tgtaactata agaccctatt attcttacac aaagcctcaa ttgtgaaagg atgagggtga 19260 gaaaggaaaa aagaaaggag gcacaaatct tcagaaattc ccctcagagt agatgctgat 19320 acccttatta tggctccaga tttcttcttt acctgtcttt ctcctaatct ctgactttat 19380 cttcttattc tccatattgc tcacttagtt ccaatgtcac tgccttccat tatcctttaa 19440 gcacatcaaa catgctccca cttgctattc ctttctcctg gagcaatctc tctgcagata 19500 accacgtggt tgtccctcct ttccttcagt ccctgctaaa acctcacctt attagaaaag 19560 ctgattccta ccaccctcta agaaatagca atcacccctt acccactgcc cttcttcttc 19620 cccttaacct actttatttt tctttcacat tattatactg acttaaatac tataaattta 19680 ctcatttgtt tattgtcttt gtttccccta atcatatgta agccctttga gtttatctag 19740 tttattgact cctacattac ctgtgcccag taatgaatga accaggtggc aatgccaccc 19800 ataagctttt tctcagcaat gagtacataa gaagtgctca tcatgatggc catcacaggt 19860 gctcaataaa taggctaatt agaagaagtc ctacacccac ccctacctga tgttaggggc 19920 ctaccatatc tgcttttgca gccctttgtg catactgcat cccttcgtca cgtcatatgg 19980 aaatctggca ttggtccatt gtgccctctt ctccctctcc atatgtgaac tttcctcaca 20040 gggattgtac cctctagctc tgtagccact gcgcacccat gctggcaaat gctggataac 20100 cagtacatat tcgctgaaac aatgggtaaa tgaacacaca gagaaacaga aaaatggcca 20160 aaaacccaag aaagacagag agaaaataaa attaaataaa gccctagtat aatccagtct 20220 tggaataaat aacgctgact attctttcaa agccattggc actcataggg taggctacga 20280 ttatacattt tgtgtatctg aaacccttct atatttattt tcagctatct cgaagtaatc 20340 tcaagcaaaa tcagaagtga aaaaaaagta gaaatgaggg ctggcataga aggtaattta 20400 ctatctagtt acaactctag ttagattaat cagggaactc tgtggctgtt gtccttatac 20460 cttacagtga ggagcatagt ttcaaaatgc ttgtcaggat acttaaaaac tataccctat 20520 atccttgata gaaataacca aaagtagaag agggctactt ctgttagcaa ttacagcagt 20580 ttgcaaggaa gagagacttc cttctcacaa ttctaagaca ggagaccgtc tcaagataga 20640 aaatggctac aaataaatat agtaaattcc acaagaggcc atcaaactta acgtaactga 20700 agccaattat tttttctgta agtatacatg gacctctttt tccccaaggg aatcatggct 20760 ctgtttggac tttcctgtta ggaattttta aaagtaaggt tttacttctg gcatctagaa 20820 taaaacaaga caagagctta tgtattatca tgaataatat tcgtttcttc cacaataatt 20880 tatttatttc ctgttgggtt cagctctgaa atgtcagatt gttatgtggc aagttcagaa 20940 gatgattcaa gccatacaaa tgagaaatac gctttattct tagtgccaat gtctaaaaga 21000 aagcaaagta atttaaatca gtaaaaactt ttaattctaa agaagcaggc agaacttgaa 21060 atctcttcat tagctctctt actaatagta gttacgttta aaaagaaaag gtatcaggga 21120 tggtcactgc tgtatcccca gtgcctacaa tgatgcctgg ctcatcctgt atgactgatg 21180 aatatcatgt attacctaac ataatcttta taacaatcct gttcatgaag attatgggag 21240 ataatgtgtg ttattcatgc atataaactc tgtgagtgaa tattattttc tctgtcttag 21300 aagtgatgag aaaggttttt taacttggtc agtttacaaa gcaagtaagg cagagaacaa 21360 gatttgaaga taggtctttc tttttcaaat gtctctgccc ttggccaagc tgcaaaaatg 21420 ccttaagtag gtgattttga tttctgttaa ctggccatga aaagtatcat gttaactcta 21480 tattgaagta atctatttaa aggcaatttt tataatcctc atgtgtcttt actcttacag 21540 atacaaagga taatgtttcc ttgtataagc catactttga attatctcac acttaataat 21600 tcaactcggt ttccacagtg aagtggcaga ttcactatta aacttctcct attcacaggt 21660 cctacacccc atcctccatt ctttgagcac cctatcctcc ctccattctc tcctcattct 21720 ttgagaggac agtgtggcag tcctcccaga acaattatct ctgtttctgg gcagattcag 21780 agctattgct ggctggaaaa cacccacata atgaatgcac catacctgct cccaggactg 21840 ggcttgcaag accctacaga ggtggccttt cagttgcatt ataattcagg tcctaggcac 21900 agtgcttgtt ccccagtgag tttgctggta aaacaattat ttccctggtc cactcctacc 21960 ttgtagacat ccagctctcc acactgctgg tcgaagcgag tgtacagtgc attgagcatg 22020 gtgatgacct gcagcggtga gcactgggag cagatggcag tgaacccaac gatgtctgag 22080 aagagcatgg tgacattact gaacttcttg gcttgcacaa cttgcccttg ccacagctgc 22140 tgagcaacct cacagggaaa tatggagcac agaaggtcta ctgtcttttt cttctcctcc 22200 tccagggctt ggtgggcttg ctcaagggta gccttcagct tccccagcct cttcttcagg 22260 ccatcttgag ctcgggcttg ttcccctatt aagaccacat ccctcagtgc attgtgaatt 22320 gggatgtctg agaggtagag ccctcgtcct gtaaaatctt ctaatctgtc cacacagggt 22380 gaccccaaaa acaagattgc actggattca acaatgtaga tcatttggcc tttgaggtcc 22440 ataacctatg aaggaaagga taaccaattg tggtttattc aagtttccca catctttctg 22500 agacaaaaga aggggaagta gatacagtaa taaacccttt gtgtttaaac ctgctgaaaa 22560 ggaacaattt gctttcttat agttcctatt ttctatgagt tctaaaatgc gtgcagatgt 22620 ttgaagagta tattagagta tactctacac agaaatcaag atttacaatg aacaatgcaa 22680 aacgctcttg cttcaaggct ttctcactaa gcagcatgct gatgcttttg ctactatatt 22740 cttcttaatg gtctcacttg attaatctga agttctattt tttgtataat atggtagtca 22800 ggaaaatgtg tcctttcttt cttccttctt ccctccctct tacttttttc tttgttctat 22860 ttttttggct tccttctctt ttagcatgtt tctcttctct tccccacttc tcccataagt 22920 acttgctcca tctggcaggt atggaagtga aaaagcctcc aaaggtaaga tttttatctt 22980 aatttttttt tctgtcgtga gagagatggg gcttcaagtg atcatcctgg accacaagaa 23040 ggtttacttg cacatgtgtg gacaaagatg tgcatgtgtg ttcttgcagc aaaaagccaa 23100 aagcaaccca aatatccttc agtagggtac tagttaaata aagtagagct taggagaacg 23160 aggcggatgg atcacccaag gtcaggagtt caagaccagc ctggccaaca tggcgaaact 23220 ttgtctgtat aaaaaataca aaaattagcc ggatgtggtg gtgcaagcct gtaatcccag 23280 ctactcgaga ggctgaagcg gggagaactg cttgaacccc ggagttggag gttgcagtga 23340 gctgagaaca cgccactgca ctccagcctg ggcgacagag caagacgcag tctaaataaa 23400 taaaataaaa tagagcacaa ccatatgatg ggataccact aagactccaa gaaagccaaa 23460 tagacctata tctaatgaca tgggaggatg cacaaaatac actgtttcat gataaaagca 23520 gttgtaaaat agcatttttt tgttttaaac aaaaaatgta tataattagt ttttcatttt 23580 tttgctacaa taaacacctt tcaatattac tcatttttta aacatctcaa tatattttat 23640 tatttttatg atagaaaagc aataataaag ctgttttcct tttggggaac aaatggtgct 23700 tctgttaaag ttatacatgt gtgaaacttc tctcttagct ttgcttgtat tatcaactat 23760 gagttagcaa actttctttc ctgagactaa ataattacca aagctctcta ccatgagatg 23820 acagaatatg ttttaacttc cacattcggt aacaattaat caaataaatt atttgttttt 23880 ttcatgtcta ttaaatgttg attaaaaatc catttagttg cctcctacaa gaaacaagtg 23940 acttttgttt gttgtaaaac aatacctctt attattcaac ctaagtcagc tatttgataa 24000 ttgaaaacat ttttatataa acatttatca aatttcatta taaaacattc cataaagata 24060 ttaaaaggca gactgcattc aataaatatt tttggagtaa tgaatcaagt aagcaaacaa 24120 atgcagaaag aattttgttt tcagtttatt acagctttac aattctttta ccattgtttt 24180 acatatgttt tatcaaacat ttgaagtgat gcatatattt ttaccctttt gtttcttgct 24240 cttaaatatg aaatagcttt catattcaag atagtattat gttttcctta cccttgaaga 24300 ttttttcaca gagttgtccc atctcctcac tcgtacaaca aactgcatat tcaacatagt 24360 catgatcccg ctaaacgtct ggttgatttt tggagtcaga atttcaaagt attcttcaaa 24420 attaggcttt ccttgaaagt ctctcctgtt catcagcctt ctgatgccat tgccaaattg 24480 cagaattgtc atatctttgt caaacatgaa atggaatgga aatgtcttgc agaatagcga 24540 tgtgggaatc accagcgagg actggggttt gctgggggac agggatggct tggtgctttt 24600 catgtgaacg gagtacaaca agtagggctg attcacaaac tcgctgcaat cattatggaa 24660 gcagggaggc attaacgaca cttccacttc cgtttcatat aatacgtgag cagctgcctt 24720 tatgatgccg ggaagaatca gggaggtggt tctcttaggg aagaagtagt aaacatgtag 24780 aaaatcatcc tccttatcca ggcatagaat ggaggcgtcc tcaagcctgc ccctttttcc 24840 tgcttcttgg caatggctgc tctgtttcag aagggtactg aagctgttta aaaaatcttt 24900 aagggtgcct ccaaccaccc caaggatgtt ttcatcttcc tcgtaacata ttttaaaaac 24960 ctcttcacca agagattctt tgataacctc cactggaact cctgaaatca cataatacat 25020 gttctgccat atcaaatatg ccaccttggt aaagctgtgc cattattcct attttcaact 25080 tgcgttattt gctgatttag ctccctttat atctgctaat aacatttcca accaaaatga 25140 aacccctata tccaaattat ctacctaggt tagcttctaa aattctagta actgaagaaa 25200 aatcccaaac tagttcatta aaaaagaatg accggttgag tataatacac aactttttat 25260 ctccctgaaa cctaactaca aaagaataat ttgaattcca gtattaagta cattttggca 25320 aaagtcctcc aatctttctg ttaacaacaa aagcaatcca tcctcatgaa gaggatcttc 25380 ctctatgagg gtttccagca gcaaaagggt cagacacgga agttaattta aaattactgg 25440 catgaaactc agttttgtct agaaatgatt acaaagcatt ttttcctgaa tcaaatcgca 25500 ttcttgttgt ttagtgttag ctgaatgaat gtctgcttgg agtatataat ttactttcta 25560 tagcattttc acgagtgcta taaggttaga aataagcctc tgtatgtctg tgtcttttta 25620 caatgttagg cttttattaa tattattttt tatcataaaa gccatgagtt acatgaagtt 25680 tttaaggagg caattttttt tttcttcttt gagacagagt ctcgctctgt cgcccaggct 25740 ggagtgcagt ggtgcgatct cggctcactg taagctctgc ctcccaggtt taggtgattc 25800 ttatgcctca cctccagagt agctgggact acaggtgcac acgagcatgc ctgcctaatt 25860 tttgtacttt tagtagagac gggttttcac catgttggcc aggctggtct tgaactcctg 25920 acctcaggtg atctgcccac ctcggcctcc taaagtgctg ggattatagg cgtgaaccac 25980 catgccttgc caagaggcag tttttttagt actttctgtt tacttgtttc ttagagctgc 26040 aggcacacag gctcaagtcg ctccacaaat cagttagtat tgtaaacgat acataatagt 26100 atgcttaatt aatatagaaa atataaatgt tataggttaa atattttgta acaaagtaac 26160 acttaacatc aaaaggaaaa agagatagga gaaagaatta acaaaggagg gtgggtatgg 26220 tgaagaagac aaaaggagtc ttggtttggg tcaggccgtc ttataagaaa gactctttga 26280 gatggcagag cctttggtgg cagatgtcca gtttttatca cgagtgaatg taagaaggtg 26340 tcaggacgac cgttttgagt tgttgaaggt ttaatttttt atagttacag agtcctctgg 26400 tgagaactga tagtggaaaa gtgtgtttgt gtttttatct tgttgtatgt agttttcatt 26460 tttttaaatt tgtttattaa ataaaacatg ctatttttgt tggcaaaatg ccctacaaaa 26520 tacaaaatgg agtctttttt aaagatggag ttagttatgt caagggtgct ctgtatgatc 26580 ttccctttct ataatctcct ttaatcttta cactctgagt accacatttt ttcttatctg 26640 taatgtggga aaaagaatag tgccctcttc atagcactgt tgtggggctt atattcagaa 26700 tagggccttg ctcctagggg ggcctcaata tacgttagtc attattatta ttatcaatat 26760 cttatacttt tagaaagatg caacctcgtg gttcatgtac tgaaactttg gaaaggacag 26820 caagacaaag tcacatatca attttttctc tcaaactaat cattaatgtg agaatactgt 26880 taactttttt cccatgactg ctcctaacct ttaaaagcat tctttatttt accttgttga 26940 gtagaatttt tgatgtataa atgaggaaca gttagttagg ttcaagatca aagcctgtga 27000 atatctgtga gacagcttcc catctagcct tatctactcc tcagcaagta aacataaaca 27060 gagaaaatgt tttaggtcaa acaaaaacat atagtatcaa aaagagttac tctttacgtt 27120 aacagaatac ataatattga acacttcata ctgggagtca ccaacaaata aatatgaata 27180 tttttggaga aaatgtgagt tctacaggta tatggcatac tttctaatta cataaacaat 27240 tctattacct gctgcaactg cttgctctgc aattgttttt tcaaagtctt ctctttccaa 27300 agatttcctg gaaatatttc aatattttaa gttataaact tataaataca tgctaaaagt 27360 tcagattata atatacagaa acaatgatag aaaatagttt aaattaggcc aggcacagcg 27420 gctcacgcct gtaatcccag cactttggga ggccgaggtg ggtggatcac aaggtcagga 27480 gttcaagagc agcctggcca atatggtgaa accccatctc tgctaaaaat acaaaaatta 27540 gctgggcatg gtggcgtgtg cctataatcc cagctacttg ggaggctgag gcagaagaat 27600 tacttgaacc gggacctggc ggggcagagg tagcagtgag ccgagatcag ccaccactgc 27660 actctagcct ggccaacaca gggaaactgt ctcaaaaaaa aaagaaagaa agaaagaaag 27720 aaaatagttt aaattccctt ttctgtagaa gcaaatcttg actatcaatt ttatgagaaa 27780 ggagcaatga catataccat ccagaacaat gttggaaaac cccaaaatat atatgacata 27840 gaatgttttg ttattcttca taagaaatat ttacctttaa agtgaacttt acatagacta 27900 atacagacag tctcagactt aggatttttt ttttttttac ctttatatcg gtgcaaaagc 27960 tgtaagtatt cagtggaaac tgtacttcaa agtttgaatt ttgattttct cccatgctac 28020 aggtatggag tacaatgctc tcatgtgatg ctgggcagtg gcagtgagcc acagctctca 28080 tgtggccatg aaattgtgag ggtaaacaac cggtactcta cagtgcactg tgttgccaga 28140 tgattttgcc taactctatg ctactttaag ttgctgagga catgtaaggt aggcaaggct 28200 aagctattca gtaggttaag cgtgttaaat gcattttcca cttagggtgt tttcaagtta 28260 cgatgggttt atctggacgt aactccactg taggtcaagg agactctgta ctcaggagaa 28320 ccttccctgg ctagcaacct caaatgtgaa gtggccaaga agaggatggt aattctgata 28380 accaagaaaa acctacatac agatagaatt cnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 28440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 28500 nnnnnnnnnn naaaccccgt ctctactaaa aatactaaaa aaatagccag gcgtggtggc 28560 gggcgcctat aatcccagct actcgggagg ctgaggcaga agaatcgctt gaacccggga 28620 ggcacaggtt gcagtgagcc gaaatttcgc cactgcactc cagtctggtt gacagggtga 28680 gactccatta tcaaaaaaat agaaaaaaaa aaaaaaaata aagcaaaaca aaacgaaaaa 28740 caaaaaaaaa cacagcatct atactgagtg aacgtatatg catattgtat gcactgttgg 28800 ttttattttt atttaaccag actgagaaag aggtaggttc atagaaactt cagaaagaaa 28860 cacaaattag gacacacttg gaaactacta ttactgtggt attagaatct ttggcatggg 28920 agattgatgg ctttagcaaa ctgtatgaaa ttcaagtatt tacttaatta tatggttcac 28980 tttataaaat atttaaattc tcttctgacc atttattcaa gtgcttcaat atgaacttct 29040 gaaatatttg cattttatag gtatatttta tgtgtttaat tttttcaaaa gcactgtttt 29100 gacttctcga gggattctca cattaagttt atcaggtcca gagataccag acaatttgaa 29160 ccacatggtt aatactagat catgtcataa agtgatatac caatgagaag ttaaaaatat 29220 gattagaaaa gctatttgtt ttgccagttt gatcaaagca aaaaaattag aattgaataa 29280 ttaattcata agtttaggat acatataatt caatttctct agttaaatag aaacagagta 29340 gaacatcaaa agtaggcctg tcatttaagg ttgactctct tgccttgtct atgagttagg 29400 gaaatcaacc ttaatgtggt aaaattttca aattataaat aaggatacta atatttaacc 29460 agtatagtaa gttattctga cagtgaggta gtaacatccc agttttgcta caaaatatat 29520 ctaaaagaat atatacaata gcatctatag ttattgtgat ttgtttttaa aaattgcaga 29580 gaatgcgaca gggtactcct gaaaaaaaaa aaagaaaaga aaaatgaaca acattttgtt 29640 tttatttagc tgtaactgaa gaaatgttgt cccaaacttt ttctgttttg ggtctggttg 29700 cagttgagat cgagttttcc ctttgtaaga gggctcattg cttgcaattt cttaacccaa 29760 aaagaagagg aaaatctata ctttaataat gtatttgcct gtgaataata ctttagaaaa 29820 gtaggatttt tttacattat agatattagc tcacaattac gattacttct ttagaatgaa 29880 ttcctagatt tgcaattaaa tcaaagaatc cacaaaattt taaggaattt ttatgcctat 29940 tgtcaattta tccttcagaa agattaatca aaataccccc tcatcagtgg tatttgagat 30000 gcctcttttt cagtacctta cccaatttaa tattgtaact tttttttctt tttttgagac 30060 agagtctcgt tctgtcgccc aggctggaat gcaatggcat gatctcggct cactacaacc 30120 tctgcctccc caggttcaag caattctact gcctcagctt cccgagtagc tgggattata 30180 ggcacccacc actgcgcctg gctaattttt gtatttttag tagagatggg gttttgccat 30240 gttggccagg ctggtttcaa actcctgacc tcaggtgacc tgtccgcccc accctcccaa 30300 agtgctagga ttacaggcgt gagccactgc acctggcctg gtattgtaac tttatgtatt 30360 tgatagttga tggttaaaaa tgacattgca tttatttttg aatatgaatc tctatcattt 30420 tatttcccat tgttttaaaa tagtattgta atcttatttg atagtgctag cttgctttta 30480 gctggctttc aggttgttca ctatacaagt gtgcttggct aagaaggcag agcccacaag 30540 tgcttttttt ttctaattct accaaagaga ccatatagga tactggggaa ctgataccag 30600 atttgtaatt ttacaatgat tattcaatct tcaaatttta tagatcaaag tccagaaata 30660 ttttcctatt ttagacatat ggagtatttg catatatgtt ttgaattttt gtaattaatc 30720 tagtaaatgc agtattcata actcattttt tatatttagg tatttttatt aaactgaaat 30780 gaagacaatt catgccattt ccccctttga tatccaggtt attcagcaaa gaaatgaaga 30840 atgtgatcat actcaatttt taattgaaga aaaagagtca aaagaagagg atttttatga 30900 agatcttgac agatttgaag aaaatggtac ccaggaatca cgcatcagcc catatacatt 30960 ctgcaaagct tttccttttc atataatatt tgaccgggac ctagtggtca ctcagtgtgg 31020 caatgctata tacagagttc tcccccaggt aaaatgacag catacttcct tggggcctga 31080 gacaaaagcc catagaaata ctattgttac aggcagccat ccattcattt aaccctctga 31140 ctatgtatta ggtaatatgc agtttattgt aagttacaca aattgtttaa ttgtgaacta 31200 agtgtatttg agcataccac aactttccct aggacaacta tttttttata ataatcacat 31260 acagaaacag taggaatgtt atgtaagaat agtatacttg ttacagtgaa aaaaaatggg 31320 taacttttta gtaacagtca cctttcacat gtttatttat tcactgaata aatattcatt 31380 ggatatctac catctgtaaa tagctttaaa aatcaactat tgatggtgtt aagggaaaag 31440 gggagatttt aaaaagaact aaaacacaaa atgatagaga tgatgatgga ttcagtagaa 31500 aaagcatcaa aggaagcagt tgggatgatg atgagaaact gctgttgact tctatgacag 31560 atccattggt aaaatccttt atgcttactc tttttttctt aaatactaac ttttggagtt 31620 gtatatggca tacgtttaga gatacttttt gcttgtaatt aaagagactt gaaaattcag 31680 gattagccca gttggatcag gtgaaatgga agcagagtag tgtgctgtcc tggctctggt 31740 attgactcta cctgtccctt actgtatgac cttgaactta ttacttcact tctctcagcc 31800 tgtttctctt tgttctggca tgagaataga aagcatatga aaaatactta gttcattgta 31860 agcaatcaat cagtgttacc tattgttttc acttttagcc ctctagataa atattaagag 31920 agggtttgct catgtttttg gtattttaat ttcatttcaa gccatacaca tttaacataa 31980 cactgtacat tttaaaagat aaattttcat tttttctcct tctgaaaatg cattgtaaat 32040 ttatgctagc ttacatttga atattagtca tctgaatcca tatcagattt catgttcttg 32100 taactattta atgtccattt aatcactgag ttgtatagat tgagatttga gttgatagtt 32160 cagctaagtt tcccttgaat tataataatt ataattttct agttttaaat ataactgagc 32220 tgatttaatc aagcgattaa tatcaaagta taaaaactat tttaactgat gtgatactct 32280 ttgctctcta tctatatttt agctccagcc tgggaattgc agccttctgt ctgtcttctc 32340 gctggttcgt cctcatattg atattagttt ccatgggatc ctttctcaca tcaatactgt 32400 ttttgtattg agaagcaagg taatcaagat attatttcat taaatgtgag aaaggtatgt 32460 cacaaattag aagtattcag gggggaaaat tatcacattc tctgagaaag tatagagaga 32520 taaaacccag acctcaaagg aagatctatt taaaaggcaa taacattgat ttttggtttg 32580 cctgaggagc agtggaactc tatcaactga ttaaaattaa cagaggtaaa ttttagagac 32640 tgtctttctg agacggcctt gtttcacatc aaaaaaccaa catcttttac tccctgtaca 32700 gctacagtgc cttattttag aaatcagaac taaaaaggat tttttttttt gaacttcact 32760 caaaaccact tatttattct ttcttaaatt gtctattgtt cttagtagcg tttttgtggg 32820 tgaataaaag tgatgaaata ttcacacagt tgataattta gatagtgctg ggatctcacg 32880 gtttctgttc ttgggataga tacagcaaaa gtcattttcc tagagtaccc attgaatcct 32940 ttctgtattt ggaactgact gtttgcagct gctctatgaa tacagggtgg gagaatgggg 33000 aggcttggct tattctttga gagtctggta atgtatgttg gaaccatacc ggaaaatcag 33060 ggggataaaa aagattcaca aaagcttgaa ataattatgt gaagagtagc acagttactg 33120 tcctgaattc agacgggaca gtcacttttc tgcagtgttg caaggcagcc actaggctta 33180 ttacaggcag aactctgaca gaaggccctc tcctgttcta atgatgcact tattctgctt 33240 tgtggtttgg tgggaaatga gatatgcgtg tccttgttgc tgaattctta ggtagggaga 33300 aaggcattca ttaatggttg tatcttgcct tttcaaggaa ggattgttgg atgtggagaa 33360 attagaatgt gaggatgaac tgactgggac tgagatcagc tgcttacgtc tcaagggtca 33420 aatgatctac ttacctgaag cagatagcat actttttcta tgttcaccaa ggtaatcatt 33480 tttagattaa ttatagtggc tatcagtacc tatctttagc taacaaagga atgccacaat 33540 attttattcc attaaattta catattctct gaggtgtaat tagattttac agcccttgtt 33600 gtgtattact ttataacaag gataatctta tttaatattt gctaataaac tcagaagaaa 33660 tcacatactg tatgttattt gccatgtctt aatacatttg tggagtgttt atgccattac 33720 gccatgggac aaataatctg cattaagcta attctatagt tttggacctt caaaataggc 33780 attatgtaga aaagtgattt tttaaagcag ctaataggat ggcataataa atacattggt 33840 atatttgaca tcaaaaatta tcttttctat agtatgttag caaaaaatct aaaaggtctc 33900 tttctgggtt tgaactattt taaattaaaa atatttacct actctaatcc aaatacactg 33960 ttccaaatat attattcaat taaaatgtat gtaaaatgtg atttttaata aacgtcagag 34020 aaaaataaaa ctatagattg ttgttttcca atttaggaaa gcattttact aggagataca 34080 tgggaattca gaaatataaa aattatgtct aaatttgctt cagaacaata gaaaagataa 34140 ctgtggggat tattgtgaga tgatattctg tgtattcata attttgtata aacaaagtgt 34200 aggtgcatta tgtagtaaca cagtcaagaa ggcatgtttt ggtgagctat tggttaaaaa 34260 tttaatctga attccaccat gatcactttt cacttaaaaa tgtggtctta aaatttctga 34320 aaaatgtaat acactttgta acaacctcct gttatattat tactattgta ttatacatta 34380 cataacaata atatataagg ccaaataaga ataatatatt gttggcacag tagttcacac 34440 ctgtaatccc agcactttcg gaggccgagg tgggtggatc acaaggtcag gagttcaaga 34500 tcagcctggc aaacatggtg aaatcccatc tctactaaaa atacaaaaat tagccaggag 34560 tggtggtggg cacctgtaat tgcagctact agggaggctg aggcaggaga gtcgcttgaa 34620 cctgggaggc agaggttgca gtgagccgag atcatgccac tgcactcctg ggcaacagag 34680 caagacactg tctcaaaata ataataataa taataataat aataatatat cgttaatttt 34740 taagtacatt taagattaat taaatattat ttaactatga catataagaa taatatatca 34800 gataatataa ggatatggat gtcttttccc tgagaaataa atatcaggaa aataaaagaa 34860 acaaaactaa gaaacataaa cgctttttaa aaaaattatt aagctacatt gatctaaaga 34920 tgtcccatct gctatcagta agtgttttct tcttctgcct cttcacaggt tgagcttagg 34980 agaatgtcag cttctcatta ggggcaattt ctatttatgg aaagaaaggt tttcttcaga 35040 aataaaagca gaaaatttgg agtttatttg ttaattccca tatgcagaca ttggtcaccc 35100 agtgagaaaa ttgcagattg tcctatgaga catgctcagg tgtgtaggaa gatttgtact 35160 cacactacat aaacaaagat caagtaaaag tagttttact tgtaaaataa ttctaagatg 35220 attagtttga atttagaaaa aacttaatgg ttcattaaaa taatgatata gggcattttt 35280 agaaccacga gaccgttatg atttaagttt tgtaactcag tatgagcatt tttgaccatt 35340 catttttgaa cgtacaatca tgttttttct ctgctcatgg gcaggtctcg tatacctttg 35400 gccgatgatc tggcttcttt ctagatgtgt ctttttctgc aagtggattt acatatccat 35460 tttaacgaaa ttaaaagaca ccgtgtattt cattagtaaa atctttgatt gttttatttt 35520 ctttataggt atgctaattt ttgttttgct ttatgaacat tgctgctgag aatatattgc 35580 attaatccaa aattctgcaa agtcacccct tttaaagaat tttggatagg attaattcaa 35640 aattcttcaa attattaatc ccagattctc caaagacata actaaatttt ttccttttag 35700 agtaattatt attcagagtg gccagaagta atgactttgc attaaaaaaa ttcaagccaa 35760 agatataacc ttatgccatt ttaaccaatt aggttttgtt gtcgttgttg ttgttgtttg 35820 agatggagtc tcgctctgtc acccagactg gaatgcagtg gcatgatctc agctcactgc 35880 agcctcctgc tcccgggttc aagcaattct cctgcctcag cctcccgagt agctgggact 35940 acaggcgcta gccaccacac ccagctcatt tttgtatttt tggtagagac gcgtttcacc 36000 atgttagcca ggctgatctt gaactcctgg cctcaagtgg tctgcccacc tcgcctccca 36060 aagtcctagg attacagatg tgagccactg tgcccagcct caaagagtac atttttaaaa 36120 agcagttaac tttttacttg tcagattatc actaaaataa taaacattat tatgttgttg 36180 gaaaaatgac acaagttaca atgatgacgc ataaaattca gcaggattga ataatgtttc 36240 aaataaatga gaaaaatttg tcaaagtaaa aagaagacat gagagccaga agtaagaatg 36300 acacatagtt ttagatacat aaattacatg ctgaggctgg gtcataaacg tggttgggaa 36360 tttttactca ttgtttccaa aagggaaaaa taagcagtta caagattcat agaatacaga 36420 aagcaaaatg aaagtttctc aaaagaagca ttattattct tgagagagag aataaagata 36480 gatttttttc ctctccttat gatgagaaaa aatttgagaa taataaatta cagcctgtgt 36540 taaatacctc aacgtttatc atagagatgt tttctacatg atcgtttcaa tttagactga 36600 tggcaccaaa aagtgcaacc tggcaaaaat gcatctcaag ttcactgctt tttggtagat 36660 gcttaaatat ctaagagaat aaactcacaa tcttcgttca attctccata attctattgt 36720 cttcagcctg atgttttgaa tggtaggagg tggcaggatc agattccttg acagaaatcg 36780 ggattgcatt gtcatgtata taagttgaga ctgatcttgc ctcatttctc catatgacag 36840 tgtcatgaac ctggacgatt tgacaaggag agggctgtat ctaagtgaca tccctctgca 36900 tgatgccacg cgcgatcttg ttcttttggg agaacaattt agagaggaat acaaactcac 36960 ccaagaactg gaaatcctca ctgacaggct acagctcacg ttaagagccc tggaagatga 37020 aaagaaaaag acagacacgt aagaatgtaa cgcttggagc actactgtta ttcataacat 37080 aatgtgactc tactatattt aagtttgaga accagactaa aaagccatgt gacctgtaat 37140 agctctggtg tagtaaataa aatctttcac attgctttta aaaagaaatt atgcttaagg 37200 aaataaacca gtcttaatgg tagtggaaat atgtctgcta tttttagaag ccaagttgga 37260 ataatatagg ccaaaatatc ttttggaaaa tcatgagaat aactcaaatt caaatttagt 37320 tcaaaaattg aagtttaact atcatttatt cctgcattct ttcatacaag agttatatat 37380 tgaccatctt cagtatgctg ggggctgccg cgtgcagaga gctgctctgc acaatgaact 37440 taaccactgg ccattacatg tttaaaaatt tattttctgg gaccttgaat ttaggattag 37500 aaagaaatga atctgacaaa ttgtaatatt atatgtaatg acaacaatta catctatagt 37560 gacatacatt agcaaaaata tcaaaatata aaacatttct atgagtggtg gattcaatta 37620 tgaaatattg agcaaggaca tgaaagataa tacttgattt tcaagaaatg tatttaagta 37680 agtacatagc atgcatacta cttggccttg ctttcatatg ctaattaaac gcacatcact 37740 gacatttata agctatttta cccaggttct tatgtgttcg ttttttaatg tttatataaa 37800 catttggcgg agcttctaag accttaggac taacgttcaa agtcacatgg gtttgtaggt 37860 ttgagttact caatattctg ttctggctgt ttgtattaga aaatcatcta ggtttcattt 37920 tttttgtatt gtgactcaat ttgctaatat ctacattcat gataaataga taaataacca 37980 gcactataaa taacatagcc tttaaattat gacatatgca gatattctgt actgaaaaag 38040 ctcttaattc acttcttttc ctaaattgtc agcttaagca gagtattagc ttctcttcct 38100 agaccaggag tccaaaactg ggagttgctg ggagttaaac agcagccttg tctttaatac 38160 gagtagctga taagtactta ttacagaact acttctttgt ttggattccc caaggtgggt 38220 ttcacattca ggaactattt gcaccatttc gagttactgt gcttcaaaaa tccttatatt 38280 attttttaga ttattagatt tagaatttgg tttttgtaga tatttgggtt tcaaggtgat 38340 gtcattagaa accatttact attaattttc tttaaagcta agtagtttta ctgttcatac 38400 ttactattca aatatagctt taatatttca taaaacattc aatgtttaaa agttgaaaat 38460 gaaagaccgg agaaattcta ctatgactag gaaaaacatt gagagctcag ctccattttg 38520 catatttaaa cttcccacat atgccctttt gctttagagc aaaaggcagg aagaaaaagg 38580 caggagggct tcatttgttc ctcttcagtc ttccaatcca gagcccatag gtctagtttc 38640 tgcttctttc cctctaaatt caccagctgt catcctcctc ctgtgtgctc acatttgtcc 38700 cttagcatga tcccatagta gtctcagttg tacctaaaga aatacaataa tttattttct 38760 agaccaactt cctctggttt tatcagtttc ttctatggag gagtgaggca tgaatgagac 38820 tggagagaaa ttctgcttct tttaatatta cttaccaaaa atagagacat atttcttatt 38880 ctaagaggca aacctgtttc cattattgtg ctattcatac catgttttag gtgctggtaa 38940 tgacagtctt catactgtga ctatcgtaat agcagggtca gaatacagga ctacaaatga 39000 aaactttgat tttctattac cagtcttttt gttttgtctg tttgcttggc ttattttgat 39060 ttggtttgaa ctagtttggg cttgcggatg tcttatgtga ttaggatgaa caaataattt 39120 atgttttctg taaatccttg cttataaata cagctaatat ttgatgctaa ttttagagga 39180 tgtcctgcta gaatccaggc ctttaaagta caaacgacac tgatgctgtg tgaaaaggac 39240 agcagaagca ctaaaggctt tcccagtatt tcttacagtg gctttctgct gatcccactg 39300 aacagattgc tgtattctgt ccttcctccg tctgttgcca atgagctgcg gcacaagcgt 39360 ccagtgcctg ccaaaagata tgacaatgtg accatcctct ttagtggcat tgtgggcttc 39420 aatgctttct gtagcaagca tgcatctgga gaaggagcca tgaagatcgt caacctcctc 39480 aacgacctct acaccagatt tgacacactg actgattccc ggaaaaaccc atttgtttat 39540 aaggcaagtg ttctttatcg ctgactgcag agctatccag aggctggcgt tctgagactc 39600 ccctccagag gccatgtcat cacagctctc tgactccagc actgcagcct tgagtacagt 39660 gagcctccat gtattcactc tttaccatgt tcttaaataa ttgcctcttg ttataaaact 39720 gtctcttcct tgtaaccaca atgaatgttt catgaagtgg gtgattccct ggttaaaatg 39780 aaatgttcac catcttattt gcacttaggc taacaaatct ggacaggctg tttatcacat 39840 gtagtataaa catagcattc attttagtcc tctgcaagag acatttttac tgagatatat 39900 aaatgctttg tacagtaaag aagtcatgaa gcatgagaag gactcagatt tgcgtttaga 39960 taacatcaac ttgaagaaga taaacattta taaggcgttt ttgccctttg tttcatttag 40020 aaataaatat aatattggaa aaaagaagtc agtggaatat tgctacataa aggcaagaat 40080 gttaagtaaa ccatctctag atatctgggg taatataact acacaaggac tatttatttt 40140 atagtcattt tctgaggtac actctagcat gtggtgagca catcatacaa acaaatgatt 40200 aatgaccgtg ataaatcact gaacttgaaa tggtgggtgt cgctgccggc aagccatcac 40260 tgttttaggt nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 40320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn agcttaaagt 40380 tgctggtaga cttgaaaaaa tatgtttttg ttagggtaat aaatgaaaca gtctttttta 40440 tgctaaccgt gaacatctaa atatatgtac tggtaggtgg agactgttgg tgacaagtat 40500 atgacagtga gtggtttacc agagccatgc attcaccatg cacgatccat ctgccacctg 40560 gccttggaca tgatggaaat tgctggccag gttcaagtag atggtgaatc tgttcaggtt 40620 agtaaatgaa gtagatattg taataatggt atgtaaacct ttttggacaa ttgattcata 40680 tcgttgtccg gaaaatcatt aacgtgtata aagaagggca tcttgacaac aaagactata 40740 gcatttcatc cagcccactg tactacattc tttcatagac atggagattt atctacctgt 40800 ttgaaattat ctagtccgtt tcatgtactg aaagtcatgg ggatgagaaa aaaacctctt 40860 gtaccttcta gtttattttt taaaacctct ttcatgtctc atgtaagtta ctagattcac 40920 atctttgaaa tcttcagact tcaaatgttt ctcttgagat aggatatttc cctggggaga 40980 tagagatttc atggagggct tttgaagttg tccataatat gcttgaaatt cttatagaca 41040 tctcttacga tgttatatca tgactctaca ttattctgta atataaagta agcaaaaatt 41100 catgtgttac aggaacagaa aaccaaacac cacatgttct cactcataag ttggagctga 41160 acaatgagaa cacatggaca caaggagggg aacatcacac accgggacct gttgggagtt 41220 tgggggcaag gggagggaga gcattaggac aaatatctaa tacgtgcggg gcttaaaacc 41280 tagataatgg gttgataggt gcagcaaacc accatggcac atatatacct atgtaacaaa 41340 cctgcacctt ctgcacctgt atgccagaac ttaaagtaaa ataaaaaaaa aaaaattcat 41400 gtgttagtga tcaaaatgat tgaagcaaag ctttcttttt tgcagataac aatagggata 41460 cacactggag aggtagttac aggtgtcata ggacagcgga tgcctcgata ctgtcttttt 41520 gggaatactg tcaacctcac aagccgaaca gaaaccacag gagaaaaggg aaaaataaat 41580 gtgtctgaat atacatacag gtgagagaaa atgtcttggt atttactgat ttgcaaagaa 41640 aatgtgtctt tgcatgtggt ttaattctct gagagatttt gttgctttaa cagagttatc 41700 accttcactc ttccccactg cttgtagagt tgtttacttt ttatttgaga agattgtgtc 41760 attatttcca ttttatatga tttcacatca tcttaggaaa tcctgatatg atcattgttt 41820 ctaaagcttt gagttacctt tcagtacaac ttcagcattt gttcttgcca tcttggttta 41880 taacatgata catgtcacct tctctataaa acttgtgtat acttctctct ctactcccct 41940 tcccttgtct tttagatgtc ttatgtctcc agaaaattca gatccacaat tccacttgga 42000 gcacagaggc ccagtgtcca tgaagggcaa aaaagaacca atgcaagttt ggtttctatc 42060 cagaaaaaat acaggaacag aggtatgact aatcaaagtg taatttgcgt acttaagaca 42120 gagtataagt atggaaaact aaagcaaaat cactgaaaat gtatttcatt tgaaaagaat 42180 tcttgctaac agaaattaac ttctaagtaa agcagtggaa ataattgaac attaatgagc 42240 caattacaat cataagtact ttttagggga tactttagtg atttttgtcc tctaatgaaa 42300 tgaaaaccaa cagactggat tgagaaggct gctttttttc ttgctgttgt ggttgttttg 42360 ctcttcctct gtagtctggc tcaaaggtaa gtgggctgtg aggggctccc ctgacccctt 42420 tcctcagctc tctaagttgg ctctgagatc accacagata agggctgtgg ttcatgggag 42480 gtttcttaac catgaagctt aagatccttt taaggggcag gctatctttc tatggagtga 42540 ttggggaatt ttggaaatcc tagtccctca tgtgtttctc ctagatagat gagtgatttt 42600 taacttgcta gaatctgatt aattccttag ggaagattta agttctttct actactatgg 42660 ttatactcca ggcagagccc atgtgctaga atggacttca gaacagtggc acatccaaag 42720 tggaatcaca cctctcatag aggccttgca cacactaaga tcatggctag cctcttccta 42780 gccttttgta aggagtggca tcagaagcaa gtttgatact ttgttaccct gttgatgtgt 42840 tgtttcctca tgggggataa gactgcatgc tcatatttct cttttagtgt gtataaactc 42900 tctaaagatg cttttagggc ataacaactt ctaaagggaa tagaatgcca gggtttgaaa 42960 tatttatctt ctttcacctg gtttaacatt aaatcatttt cctatgaggt ggacatgctt 43020 attgatgttc ttctgataaa ctaagccgta attacctcaa acattggtca tcttagtcgt 43080 gcggtcctgt ttttcccaga attatggtgg gaatgattta cagaaactgt agatgtgaac 43140 gtggatcaca ctctcataaa gagaaaaata tatttaattg atattataaa cctcactatc 43200 actgtaaatc aatccctctt ttcttctgcc tatttaagga aacaaagcag gatgatgact 43260 gaatcttgga ttatggggtg aagaggagta cagactaggt tccagttttc tcctaacacg 43320 tgccaagccc aggagcagtt cttccctatg gatacagatt ttcttttgtc cttgtccatt 43380 accccaagac tttcttctag atatatctct cactatccgt tattcaacct tagctctgct 43440 ttctattact ttttaggctt tagtatatta tctaaagttt ggcttttgat gtggatgatg 43500 tgagcttcat gtgtcttaaa atctactaca agcattacct aacatggtga tctgcaagta 43560 gtaggcaccc aataaatatt tgttgaattt agttaaatga aactgaacag tgtttggcca 43620 tgtgtatatt tatatcatgt ttaccaaatc tgtttagtgt tccacatata tgtatatgta 43680 tattttaatg actataatgt aataaagttt atatcatgtt ggtgtatatc attatagaaa 43740 tcattttcta aaggagtgaa ttctaagttt taggggaaaa aatgcaattt attttcagac 43800 tcccaaagta agaattaaca tatcatgcta agaaaatagt gactattttg aagtatgcta 43860 cttccctttc agaaatatag aatacacgtt tctgttatta aagtatttga ttactaattc 43920 aaatcatatg gcaattataa ttcttctaaa atgctatcat ttgtaactgt atcccctgta 43980 ttaaatctca ttaaccacag gcagctgtta cagaaagctg cattgtttca tttttagctg 44040 ttacattagt tcaggctaaa tgttgggagc tccaaccaca tccaagaata aatctggaaa 44100 cacactgctg ggatactgct gttagagccc ttcttggcct tgtattccca gaaatgagct 44160 ccctttcctt agcttagaag aatgtgatta tatccaggac atcatgttca gaaaacttag 44220 tttactttca gcatagaatg cattactgtt ggaataattg gcctctagct cttaaatgtc 44280 tctgataact tattaatatc tatctttata aaatagagtg caactacttt tgtgtaaaaa 44340 tgtttgcctt taaatttagt atttcatatc agcacatcga tatatgtata aatgttccat 44400 gttaatgtgt aaaagagtct gtaataaatt atttttttca cgtgtctcta tacagttttt 44460 atttcaataa aaatattaac attatttttc attttattaa tgccattatt ttgtaaatac 44520 attttacaat tttgcctatt tgctaccatt ataaattttc actgactcct catagacagc 44580 atattcttaa atgctatatt tctttttaat accaacagag tgacaggaaa taaagactgg 44640 gcatggaaag gggaaacagt taaatattga atgtcatcag cttatttaaa gagctctgaa 44700 tcatttaggg atgatagaca tgtatacaag taatgaaaat atgcgatatt cataatgaag 44760 aaatatattc aaatgcagaa ataatactta gagaaagagt aacttgaaag aaaagaggtg 44820 aagtggggat agttgttatg cttattttgg ggggagagtt aggaatactc agaaactaca 44880 ttgacactgt catcttccca gaatcctgca tatatttatt tagcatctac tttgggtagg 44940 tactgctcaa gatatttgag aaatgtcatt gaacaaaaca tctctgcctt tgaatacctc 45000 atattccaat ttttacatct tgcatgtttt taaatctcta ctttattccg ccaacactct 45060 tcgaagtcca ggcactcatc atgtgtaact tgaacaattg caagatcttc cctataaatc 45120 tgtctatttc ctacctttgc cacctccatt ccacactctc aaattgctgc cagagtggtc 45180 ttttacaaat ccaaattgct ctcctgcgta caatcactta aatgctactc attgtctgta 45240 caccttagca aagcatgtaa acattgtcac cttaaccagc cttatctgct agttgctacc 45300 tttgtaattc tatgctctgc tctgtctgaa taccctctac ccatactctg ctgcaaattc 45360 atttcttgct aagactcagc tcaatatcct cttgttggaa atttccctgt taggaaagtt 45420 tagcttcccc tattgtccca ggatctttac aatggttcac atattatctg tttatatata 45480 ggattcctta ttaggcttta agtttcttat ggctaaatta gttcctctta caccagatat 45540 tccttgaacc ttccacaaaa taggcacttg acaaatattt gttgaacaaa ggaatggagg 45600 gagcagcctc ttcgagcagt ggggctagaa gccagattta aagggaagga ggaataagaa 45660 atgagataga ttggagaaaa gaggaggcac aaaaatggca ctgttaaact tgtgcttaga 45720 gaaaatcatg ctgacattgg tgcaggggct aatttagagg gaaagagcct ggtttaaggt 45780 tggcattagg gggctactgt acttcatgca taggcctgct ttaaggtgat ggcaggacag 45840 gaggctaatt gaagagcagt caagattcac attaaaagtg cataataacg cacaggccaa 45900 aggggatgag aaggttaggg ccaagtagaa agcttctggc ttgcttagtg ggtagttgat 45960 ggttccatta cacgtggtgg aggatgtaga aaggtaactg agcttagagg aaaggtgcct 46020 aatattaggc atggctttga agttctggtg aatacatttt taaaaaacaa tgattttcag 46080 gtagcattaa tgcgaggaga gaatagaact tttttttcca agttgggcca aactgctttg 46140 aatttccaaa caacctgtta ctagctccat gatattggta aagtcattta acctttgtgt 46200 aacctggtta ggtcattgta aagacagaga ttataataac cactcactag agttgttggc 46260 agatcaaact gaaatacgaa gaagtcagag gagtgggact tttggcttgc catttcagaa 46320 agattaaaag gtatttaaga aatgaaaact tccaaatctt tggtaacaga tattgttata 46380 gttgaatcat ggacagcaca tgtcatgaaa aaacaggagg gtcaacaaga aaaatcaata 46440 aacacattta tctttgccaa ttattgaaac aaaccccagt ggaaaccacc aaaaaattag 46500 aaccaaatgt tgtgaaacga taacagttta tcagtttgaa ctgaattgtt ttaaacttta 46560 acaattaaat accattttaa aggtatgtat ttttaattat ataagttata tatttattat 46620 attatagatt acatatatta tatgtatttt agcactaatt cactatttca ttcagtttaa 46680 atctgtattt gattcaccaa atgatatgtt ctatttcctt tgtcattgaa aggaaaaata 46740 atttgcttac ctccctcata ggttttcttt cttttttttt tgtaccaccc tcactaatgg 46800 tttatagaac ctctagaggg cagcagagaa tgctgtttgt aattgaaatc tgaaaaagaa 46860 ctgaaaggag acattctctc acaggacaga gttcatcttg tctcctagct gtcacttcct 46920 gtgtgcaacc ttgaattcac tccttccact ctttctcaag ttcaaaggaa tataataaaa 46980 ataattattt ctactcatta agaccatttg tatagcatga aatctatcca cacgtatctt 47040 gctctgaaat gtcaccaaaa tatttcctgc taattctgca ggaaggtaaa aacatttgct 47100 aaaacaagca atttgtaata ggaacaatag ttttgtacaa tatttttatg agtaaactaa 47160 cttgcttgta gctcatgtta aaagttatta aatagaaata ggatactatt tacttataag 47220 gataaaattg gaaaaaaatt tattttacat taatagaaaa tgcttttgtg attcttcctt 47280 gctacttata ccgtatcaaa gagaagaaat atagtgtgag ttaacaaaac aaaatttaac 47340 aaaaaacaaa gcaccgtaca gagaatcaag aacctgagtc caattctggt gaggaaatct 47400 tgtggccttt acaagtcact taactttaca actcagttac ctcatctgaa aaaaaggagc 47460 tggttaaggt gatatccaaa ctaaattatt gctccaaaat tccatcattt cgttggtaac 47520 aattccaggg tttttgtatc agaagcattc taaccaaagt gactccatct ttacagaggc 47580 tgggtaaaat gaggctgaga cctgctgggc tgcgttccca ggacgttggg cattcttagt 47640 cacagaatgt ctacagttaa gggaataggt taataatgtt taccaaacag acccaggact 47700 taacagaccc aggaaatgtc caggtgtccc aatatcttag gaataaatgt attcttaatt 47760 taagaatacg ttttgcttta aagataataa tacagattat tgtagaacag tagttaagca 47820 aatactcagc tgataaaaca ccatgcagta aagaagcagg ctgaaaccca ccaaaaccaa 47880 gatggcagtg aaagccacct ctggtcatct tcactgctca ttatatccca attataataa 47940 attagcagtt taaaagatgc tcctactagc gccatggcag tttacagatg ccatggcaac 48000 atcagaagtt actctatctg gtctgaaagg gggagtaact ctcagtcctg ggaattctcc 48060 actcctttcc tggaaaactc atgaatattc cacccctcgt ttagcattta atcaagaaat 48120 aatcataagt ttactcagtc cagcagccat gccactgctc tatggggtag ccattctttt 48180 actcctttac tttcctaata aagttgtttt cactgtactc tgtggattca cctcagattc 48240 tttcttggat gagatccaag aactctctct tgtggtctgg atcagtaccc ttttccagta 48300 actcttgtgc taggattttg gcctgaaact tttgggaata caatctaaag ttttgacaat 48360 ctagtagtgt tgaatagatc aaccgaaaga ctgaaagcta acttccaaga aataaacaaa 48420 tctgaggtaa acacttggtg aggacttttg actccaagct aaaatcaaaa tgactaaggt 48480 cctgattagt gtatatgagt attaatgtta tgtatgtcta ctaaaaatgt gccctataat 48540 atttaattat gttagctaaa tttaagtgtt taataaacag tcacattctt gatgacctga 48600 aaacaaggag agaatgtatg tctctatttc aattatatgt actcacacaa ttgctgtggt 48660 acagtctatc ccaaataggg cttaaatata ggggccagtc atggtggctc acgcctgtaa 48720 accagcactt tgggaggcca aggcaggtga atcacttgag gtcgggagtt tgagaccagc 48780 ctagtcaaca tggtgaaaat gtctctacag aaaatacaaa aattagccgg atgttgtgat 48840 gggcacctgt aatcccagat actcgggagg ctgaggcagc acaatcactt gagcctggga 48900 ggcggaggtt gcagtgggct gagatcacta ctgcactcca gcctgggcgt cagagcaaga 48960 atctgtctca aaaaaaatta caggtaaaaa atatcagtga gtattacata taggttgggg 49020 tttttaaaat gttattctta tgaaatatat ttcccaatta tgtaaagtag tgaccacagt 49080 tgcctaccaa acagttttag atcattaggg ggaaaggagt cacataaaat taaagtatta 49140 acttagaaaa cacttttaaa ctgttgtaaa tactgtccct tttgtggttt tctgcatcac 49200 atgaataaat gtcaatatat taataatttt tatataccca tgaatataat accatcatat 49260 tgttctggac aacaacagta cctaaatctg acactgtgac tacatacagt tttagatgta 49320 ctagtacaaa taacgtaatc aaagatattt tgtttatttt tgtgtaaaaa tcattacagt 49380 ctaaggcagg catgggtgaa atcagcgact gagtcctctg ctcccgggca actggggctc 49440 tgaagagtgg gggtcagggg agtcccctga gaagtttgtg tcctggaatc caagacaagg 49500 tgagggcagg gcctgaagaa aatacaagtc tcaggaaata tgtagatttc aaattgatct 49560 gacaaaaaac cctagtggtc tgtccaaaga agccagtagc cagaagaagc attacaggca 49620 ggaagcatta tgaagttaag tgtgtctggt gttggaagcc ttttgcacag tgctagtttg 49680 tgtgtgcagt ggtgattttg gcacctgttt aataattgta tatcctcaat cgcaagtatt 49740 ggacaggata ttgaataatg ggaggttgca atttcttaat gtgttagtat aactattttt 49800 tcctctaaag cacagtgttt ttaagtctgt ttagaaaact tccaggcgcg gtggctcatg 49860 cctgtaatcc cagcactttg ggaggccgag gtgggcggat cacgagatca agagtttgat 49920 accagcctga ccaacatgtt gaaaccccgt ctctactaaa aatacaaaaa ttagctgagc 49980 gtggtgtcac gtgcctgtaa tccaagctac tcaggaggct gaggcaggat aatggcttga 50040 acccaggagg aggagattgc agtgagctgg gatagcgcta ctgcactcca gcttgggcga 50100 cagagcgaaa ctccatctca aaaaaaaaaa aagaacactg caagggtaat gtctaacagc 50160 atggtttaat agtaatacaa tgtgaggccc aaatataagt cacatatgac atggattttt 50220 aaatttaaaa atttctagta gccacacttc aaaaggtaga aaccagtgaa attgacttta 50280 ataatatgtt ttatttaact cagtataagc aaaatatgat catttcaaca tgtaatacat 50340 ttaaaatcat taattaaata tttaaaatca gtgtgtattt taaaatcagt gtgtatttat 50400 acactttaaa atcagtgcgt atttatacac tttaaaatca gtgcgtattt atacactaaa 50460 atcagtgcgt atttctacac tttaaaatca gtgtgtattt atacactaaa atcagtgcgt 50520 atttatacat tttaaaatca gtgtgtattt atacacttta aaatcagtgt gtatttatac 50580 actaaaatca gtgtgtattt atacacttta aaatcagtgt gtatttatac actaaaatca 50640 gtgtgtattt atacacttta aaatcagtgt gtatttatac actaaaatca gtgtgtattt 50700 atacacttta aaatcagtgt gtatttatac actttaaaat cagtgcatat ttatacacta 50760 aaatcagtgc gtatttctac actttaaaat cagtgtgtat ttatacacta aaatcagtgc 50820 gtatttatac attttaaaat cagtgtgtat ttatacactt taaaatcagt gtgtatttat 50880 acactaaaat cagtgtgtat tttacatttt gcagcacatc taatttcaga ccagccactt 50940 ttcaagtgct cagtggtctc atgaagctag tggctatcat attgaacagt gcaggtctac 51000 atatacaggt tattgattgg atcttttatc caaaacaatc atcttagata cttgattgtt 51060 ttgtttcctc cttaccagaa gattggatca accttgttat ttataaaaac aaaacaagta 51120 gctgagtagt tgaaggggtc attaaattaa gaaaaaagag tataggaagg tttatacttt 51180 ttattctgat ttataagatg tagttttgaa tagagtgttt cttttgaact ctaacattct 51240 atatactaca acataatata aatggtgata aaaagagtaa taaaggaaaa tgagttctat 51300 atgaggtatt tctcaaacaa catttgcaga aaagaaagtg tactatttta taagttgcta 51360 acattagcaa cttcatgatt tgagatgata aaggatctta catgttcacc tgacaattct 51420 tttgcagtct tttaataaga agcaattata aagcactaag aagcacctat tatcttttga 51480 caaagttaag gctctttata aagatctagt tcatcagtca gttaccaccc acatcatatt 51540 tctttttttc atttgtattc ctgttaaagg ttctttccct gtcaacccaa cttactcttt 51600 ttgcacttga cagtaatatt accaaagtat taactgaaag agaattatgc aaaagttgac 51660 taccaaaaga tttgtaccct tgaatacaca ggaacaattt tcacttgatt tgtgctcagt 51720 aaagtgtatc agaaagttca gaacccttca gacctcagga ggagcaaatg ttcctgtcac 51780 actgaccata tgtctttttg tgaatttacc aaaagttctt cctgattcat gaatgtttga 51840 aattcaaaac tcattgtata cagcttgttg gatttcttga catgtaaagc acttttaaaa 51900 agagttcttt taaagatagt gttaatacta taataaaagt tataaaattt gcttttaaaa 51960 tatatatttc tttaaatatt gtgctgtatt ataactgacc atcaaacaac agacatgaga 52020 gttgagataa gtggtgacca gatccctgct gccctcaggc tcacaactac cagcatggaa 52080 atcagggagc aatcctgcag tcactgcaag ttcaatggtg atttgaacgt tttacatgtt 52140 taatccttgc agcaatttat taagtatata ctcttattat cgactccatt ttactcatga 52200 ggaaactgga gcacataaag tttggggaac tgcccaaatg ttaaacagag agtgagtggg 52260 taaagccagt atccataccc agacaaatgg attccagagc ccaagcttcc aactaggatg 52320 ctcggatgca tctcatagca aaataagcaa gcggctggca agtggcgtac atctggtggg 52380 cgttttaaca ccagatgagt gtgtgtcagg taacaaagtt ctagtcaggc aagcaggcaa 52440 ggagtgtgaa gaagatatgg caggtggtat tggccctaaa ctttacgact aactctgaac 52500 aacaggatgt ggaaggctgg ggcacaagaa gtgggggctg aggcagggaa ggcaaacatg 52560 ttcccctaaa gatggatcac aagacagcga ttataaaggc tagaccggga gaacctatat 52620 gttgtgcagg aggaaaaaca gcctgatgct aattcgcccc tctgtgtata aaaggcttac 52680 gttcttctgc ctgataccca gattggttct agagttttca gatttgggga ctgatgaact 52740 ccaacagcaa accctgatga gctgtacaag tagggaaagc atgtccttga ctgtatcctc 52800 ccattaaggc atctgctaag agagcagtct tgctggatgt ggaaggaatc ctccataaag 52860 gtagtcagcc ttagtcttcc tcactctgcc accatgacta gcatttcaat cttatttgta 52920 tgggcttctg acaaaggaaa tttggggcag ggagggggct ctttctaagg cttataacag 52980 gttcttccag aaatatagaa gaaatttttc agctttccca atctcagtta ggtctgtgct 53040 tccatagcat ctgagtaaaa gaccattgct attcttctat ctgttacaat tttaatgatt 53100 tactgtcttc tggtcaaata gactgtggga gaaattaggt cagacactgc acatccactc 53160 attggctcca gtagccagtt ccagagcctt gtcacagagt aggggttcag aattgtcagg 53220 tatccttcct ccaaatactg gagaagagtt ggtgaagaag aatgtcgttt gtacaacttg 53280 taaagtatct gtgttttcca cttgatggtc tgttttgtgc attggctgta gatttattta 53340 ctggaacggc cttctcatca tatacatgtg cacatccatg actgatgcca tgctgtagtt 53400 tttaacaaag cattgttgaa catttgaagg ataatatcag aagggaatag ctgcgcagaa 53460 tataaaacac tcaagattaa aaattcttct gcctgcaatc ccagcacttt gggaggctga 53520 ggtgggcaga tcacaaggtc aaaaaatgga gaccatcctg gccaaatggt gaaaccccat 53580 ctctactaaa aacacaaaaa ttagctgggc atggtggctg gtgcctgtag tcccagctac 53640 ttgagaggct gaggtaggag aatcacttga acccgggagg cagaggttgc agtgagctga 53700 gatcgcgcct ctacactcca gcctggcaac agagtgcagt ctcaaaaaaa aaaaaaaaat 53760 aaatgcttct tctcaagttt ctaaattccc gtggctatat tcaatcacat ggcgtcccag 53820 tgccagccaa aggacacctc agcgaggaat ctttctgtct tcactcttca ccaagtccca 53880 gtgcctctaa atctctcttg gcctacctaa agttatcgtc cgttttcttg cattattgat 53940 gtctagaact tatcacaatt tattatgaag ttaagggaat aatttttaaa ttgttagcaa 54000 ttatccttac caccagataa taaaacattg ttgatatata tgaagacatc ccagcattca 54060 ggcataatgt atgcactttc ttctaaatgg tgccgtaaaa gagaacgtca tttgcagata 54120 tttgccataa gcaacaacca ctctaagtga agagaaaaag taccttgccc cctctggttt 54180 tcacttagta atttaggaat tacacgttat ttatatggga ttgtatatgt ttatccacat 54240 tttaatgact agatttaaat tgattttaaa ttactatcaa tatattatga taatcattaa 54300 aacatcatta catcattaaa caatcattta gaaagttttg ctccttttaa aactaatgca 54360 tgaattaagc tgaataacaa caaaaaagta tcaatatttt accatcacta acaccattga 54420 ataatactgt tttcatgacc acaaagtacc gttttgccaa gcataagtta tttaggctgc 54480 caaattatac tgaaaacaaa tatcaaatgt caccaaaata taaaatcttc atccattttt 54540 ctatatcaga ttgcaagaga agaaagaacc tgatttgctc agtgattcat atgaatctga 54600 tttgaattaa taatgtaaaa ttattaatga cagtattgga ttatatccca tataattaag 54660 aaaacttcct gaattcatat ttatgtaagt aaatgactta aataataaat aaatgaggga 54720 taaaggtcaa ttcttcctta cagaatgatt tcaataaaaa taattatgaa cagatcagta 54780 cttggggcaa gggaaggaga gaatttggag agttggagta atggatctaa cgtggagtga 54840 aattaatcaa caggttaaac tattctatat ctttgtgaaa ttccctttac cataaaaagt 54900 ggttcgtgta tttaaaaatg gttctctgtt acaccatcct tagtataata gaaaagtttt 54960 tttttctcaa tgtattctca gggaataacc ccatgctttt agttacagta ttttccccag 55020 tttatttttt ttttgtaaaa gattacacaa tatcaagtcc ttttttcgct caatatcata 55080 gttaagcatt tttccacata gagtgagtat ttaatgttca cataaaattt catcccgtag 55140 ctggaccatg attttctcaa ccattttagc atagccagac attgacactc agaaactgag 55200 gatggattga gtagtgaggt acagcccagt tttcttgtgc atttcagttg cctacatggt 55260 acagccctaa tacactgaag caagacctac ttgttcattc agcacatatt aattgagctc 55320 ccacaacatg ccagactctc gtctaagagc ataggttaga ttgatggaaa ataaacagag 55380 ctccttgccc tcagagggct taccttctag cagaggggga cagaaaatac accataaata 55440 tattaggttg ggtcaaaagt aattgtgttt ttgccattaa aaataatttg caccaaccta 55500 ataatataac cataaattaa atgatagagg acgatgtatt ctatggggaa aaaattaagc 55560 acagaggaaa ggagatcagg agtgcaagga tggggtgggg ctacttgcaa tttggaaatt 55620 gagagttggg gctggcctca atgaagggca aagatgatga gtgagcaaac actggaatgc 55680 tgtgtggtag gtattgcaac tatttgaaag aaaagcagct cagggagaga ctgccagtat 55740 ttgggctcat gacaggttca agttacttat gttccagaaa ccaaaaggag agtgtaccag 55800 gagcaatcaa gccttttagt acctatgagt ttaattataa gaaagaaaat gtatgtccca 55860 gggaagttga gacagttcat aattatccat caatccaatg taataaggag atatttgaaa 55920 tatcctgcat gcattgttat tctcactcat gaaacctttt tatgcctttg gcagttgtct 55980 gctccctgct tcctggtaat ggtttttgat gactatgtat tatttcatca tatagcttat 56040 ccacaagttt ttcacaactc ttcttttgtt ggatatttgg attaattcca attttcaatt 56100 attataagca atgctacagt gatatccttg cttatataaa ttatttttaa agaatataaa 56160 aataataacc ggccagatgt gatgactcat gcctgtaatc ccagcaattt ggaaggctgg 56220 ggcacagggt cccttgagcc cagaagttca aggctgcagt gagctatgat cacaccactg 56280 cactccagcc tggctgacag aaagagactc tgtctctaaa taaaaccaaa aactctattt 56340 gcagttgact agttgacaat acaggaaaat ggacatactc ctaaactact actttgtatt 56400 taactgtgtg aagggtaatt tggaaataca taaaaaacac attaaatgtt atcaagaaat 56460 tcttgaaatt tattctctga aaataatatg aaatgggcag aacattttgt gattaagaat 56520 gttcattgaa ttatgatttg taataattaa aaatgaaaac aaatttaact tataaaaaac 56580 ttaatgatat atctatatat tgaattatat ggaaaaaatc tcataggtag tacattgtat 56640 atacaatatt gtatacacat tgtatattat gattttatgg catatttgta ttttttggtg 56700 tctggtggtg ggatgtttgc ttttataaac actttgaaac acatgtgtaa taaagatata 56760 agtgttctgt taatgtacac agtatttttt gtnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56880 nnnnnnnnnn nngaattctt taaagtacct aatgctttct ttaggagaga ggttatatca 56940 agttaaaagt tctcaaggaa agaactggtc tcagaatttt tgccatagcc accagccaca 57000 ggtggctatt gagcacatga aatgtggcag gttcaaatcg agatgtgagt gtaaaatata 57060 caccagattt caaacagtaa gcactaaaac agatggaaaa tagctcctga acaattctta 57120 tattgattgt ttggtcaaat gaaatctatt cttaaaatta aattttaagg ccaggcgtgg 57180 tggctcacac ctgtaatcct agcactttgg gaggcagagg caggcggatc acctgacgtc 57240 acgattgtga gatcaacctg gccaacatag tgacactcca acttcattaa aaatacaaaa 57300 attagccggg catggtggtg cacatctgta gtcccagcta cttgggaggc tgaggcaaga 57360 gaaacgcttg aacaagggag gtggaggttg cagtgagccg agattgtacc actgcacccc 57420 agcctgggca gtgagccgag atcacaccac tgtaccccag cctgggctat agagtaagac 57480 tccgtctcaa aaaaaaaaaa aaaaaaaact aaattttgct tatttctttc tacattttta 57540 caggaaaatc caaaattata tatgtgtctc gaactatatt tctactggat gtctccatcc 57600 tggacttttc tacaacaaat aactgtcact ctttataatt ttaactccct gcataccgtt 57660 ttctgcccac catctttctg tttacagctt acttgtttta ccatcctgac ctcagcaatc 57720 tttctgggat ctctgatgca ctgaactaac taacgtttct ctgtccttca ccccttgagt 57780 gtgcttgccc cactctcacc ttgttgcaca tggcgattaa tgattagtga tttcagtcac 57840 tccttgccat ttactctcag ttctcttgtt ctctctcact tggtcacact tctagagcca 57900 aaattctatc ctggataaat tgcactctcc agtgatttgg tgcctgctct ctggcaactg 57960 aatgtggctg gagaaaaaaa aataaacaaa aactcaaaac caaatttact agattcagtt 58020 taaaatcata accatgaatg tcaagtggac tctccacatt gctaggacat cacgcgatac 58080 attttgagat cattcttttg tttttgtttt tgtgttagtt ttgagacagc atcacatctc 58140 tctctgtcac acagtctgga gtacagtggc acaataatgg gtcactgcaa tctctgcctc 58200 ccaagttcaa gcgattcttg tgcctcagca tcctgagtag ctgcaactac aggtgcatgc 58260 caccacatgt ggataatttt tgtattttta ttaaaggtgg ggttttgcca tgttggccag 58320 gctgatctca aactcctggc ctcaagtgac ccacctgcct tgtcctccca aactgttagg 58380 attacaggtg tgagccactg tggctggcct actagaccat tgattttatc aattttcaca 58440 ccttagcctc tctctcaaac ctctatttct actttcagct gattactctg atatcttttt 58500 cactaagaaa tttcaaataa tcagaggaat attaccattt tgatatctat agcctcctag 58560 tatgtattcc catataatct gaattcctat gttcatatca caaatgaact atctattgca 58620 aactctctat ttgggtgtta aatcatgatt catcgaactc ctcaagaatt gtgctctgac 58680 aaataggcct tcttcctccc acatatttac tttttagggc atttatactt acgaatttat 58740 aaatttataa aaatggtatt atgtctctaa tttatgaaaa aacttttatt ggttacattt 58800 tccgtgtcgg caccatttta ttgttttgct tagtagtaaa actcctcaag acagtggttt 58860 gttatttgct tctaccactt ttctaacact gccacattgc tgaaatcaat ggtcaatatt 58920 caggcctcat tgtgcttgac ccatcagcaa catttgacac agctgatcac tatgttcatt 58980 accctttcca gcttctggta gcttttagca ttcctgagca tgcggcagca ttaacttcac 59040 ctctgcttca cctccatctt cacacagctt tcctttctgc acttttctgt gtccaagtat 59100 tcttctcttt ttggtctttt atgaagacac cagttattcg gatttaagaa ccaccttaag 59160 tccaggatct tgagatcctt aaataattat atttataaaa atcttggccg ggcacagtgg 59220 ctcacgcctg aatcccacca ctttgggagg ttgaggtggg cagatcacga ggtcaagaga 59280 tcgagaccat cctggccaac atggtgaaac cccatgtcta ctaaaaatac aaaaattagc 59340 tgggtgttgt ggcatgcaac tgtagtccca gctactcagg aggctgaggc aggagaattg 59400 cttgaacccg ggaggcagag gttgcagtga gctgagatca tgccactgca ctccagcctg 59460 gcaatagagt gagactctgt ctcaaaacaa aacaaaacaa acaaacaaaa aattatttcc 59520 aaagaaagtc acattctgag attctagatg gacatggatt ttgggaaata ctgttttact 59580 ctttgcagga ccttggtatc cttaaataac tataactata aagatcctat ttccaaacaa 59640 ggtcataatt taaagttctg gatggacatg aattttggga aatactattt tactacagtg 59700 tgtaaaatct acatgagaaa aaattgacaa tcatttatgt tatattatca cccaggaatt 59760 gtgccttttc aatgttctat tgtttatttt gctatctttg agttattttt gttttatttt 59820 gatctatttt tttagaaaat gcatttacca cacaactaat aaccttctac tagaatagtg 59880 cccaaagtta tggaaatgat gagaaatcaa tatttttgtt ctaaataatt gacagtcagg 59940 taaatgtctc cctgatgttc tagtacacag agataagaga tcagagttga gaaaacgaaa 60000 cttaaaactg cagggagaat atttgaagaa accctgtcca agtcaccttg acccatgaaa 60060 tcatgtactg tgtggacaca taatttggca agctaatggg cctacaagtt aagcattagt 60120 ggagtccttc cccatctctt gaccaataat aggaatactt tcaatattta tggcaaacca 60180 tggagttgtt ttaaactgaa gctgtcccac taatggtgtg gctgcagctg agtgacaaac 60240 agaaacctcc cacaaactga accagtaact gcccaagaaa ctggagaact gaaataagaa 60300 actcatcaac aggttcccaa aagccttttg ttttcaaaag actgaacata ccagctaggt 60360 atgggctttt taaaaagtac aaatgaggct gagcacggtg gctcacgcct gcaatcctag 60420 cactttggga ggccgaggca ggtggatcac gaggtcagga gttcaagacc agcctgacca 60480 atatggtgaa accccatctc tactaaaaat acaaaaatta gcccggtgtg gtgtcacacg 60540 cctataatcc cagctactca ggaggttgag gcaggagaat cacctgaacc caggaggcag 60600 acgttgcagt gagcggagat ggtgccactg cactccagcc tgggcaacag agtgagattc 60660 catctcaaaa aaaaaaaaaa gtacaaatga ttaggatata tccatggcac atttaataaa 60720 tgtttgttga taatgatatc acaggcatgt agtatttact tatgtatatt actcctgtgc 60780 atattatttt taaaaagagt tgcttcctat atttaaagat ctaaggatga acatttcatg 60840 caatcagtac atgaaagtaa aaatcaaaag aattgaacaa gttcaacatc ttgctagcta 60900 cggtcatgta caaaaaatgg aataaagaaa taagcagtat gtgattcact tctgttttga 60960 cccaagaagt ttaggatttt gactggtagt atatatgata gaaaagtata aaataaatat 61020 aacataagta aattcttatt taaaaagagc atccagaagc tagtatttct atatgttgcc 61080 ttaagtctct cttcataacc actcaaatta tcgagagcta taatggtcag gaactctatc 61140 tccttttcta cagctaaaca tgttggtgct ttaaattggg aaacccaatt ttaaagcatt 61200 acaagtttga gttattaaat tagcaatgtt tttttcttcc aaaatatatt tacattattt 61260 acttttaata gataattgtg gaaaatgagt gatatggttt tgcttggtcc ctacccaaat 61320 ctcatcttga attgtagctc ctgtgatttc cacatgttgg gggagggacc cagtagcagg 61380 taactgaatc atgggagcag gtctttccct tgctgttctc atgatagtga ataagtatca 61440 cgagatctga tggttttatt aaggggagct cccttgcaca tgtcctcttg cctgctgcca 61500 tgtaagatgg gactttgtca tttgccttct gccatgattg taagacatcc ccagccatgt 61560 ggaactgagt ccattaaacc tctttccttt ataaattacc tagtctcagg tatgtcatta 61620 ttagcagcgt gagaacagac taatacagta aattggtacc ggtagagtgg ggagctgcga 61680 taaagatacc caaaaacatg gaagtgactt tggaactggg taacaggcag agcttggaac 61740 agtttggaag gctcagaaga agacagggag atgtgggaaa gtttggaaca ttctagagaa 61800 ttgttgaata aaatgctgat agtgatatga acaataaagt ccaagctgag gtagtctcag 61860 atgggcatga gaaacttgtt ggaaactaga gcaaaggtga ctcttgctat gttttagcag 61920 agactggtgg cattttgccc ctgccctgga ggtctgtgga actttgaacc tgagagtgat 61980 gaattaggac atctggtgca agaaatattt ctaaggagaa aagtgttcaa gaggttactt 62040 gggtactatt aaaagtattg agttttatgt atgcacaaca atatggtttg gaattggaac 62100 ttatgtttaa aagggaagca gagcataaaa gttcagaaaa tttgcagcct gatgatgcca 62160 tagaaaagaa aaactcattt tctgaggaga aattcaagtg agctgtagaa atttgcataa 62220 gtaatgagga gtcaaatgtt aattgccaag acaatgggaa aaatgtctcc atggcatgtc 62280 agagatcttc atggcagccc ctctcatcac aagcagggag gcctaggagg aataaatggt 62340 tttatgggcc aggcccaggg ccttgctgct ttgtgcagtc tcaggacgag ccggtgggta 62400 cacagaaatc atggatgaaa ttggaaatca tcattctcag taaactatcg caagaacaaa 62460 aaaccaaaca ctgcatattc tgactcatag gtgggagttg aacaatgaga acacatggac 62520 acagcaaggg gaacatcaca ctctggggac tgttgtgggg tggggggagg gggagggata 62580 gcattgggag atatacctaa tgctagatga cgagttagtt ggtgcagcgc accagcatgg 62640 cacatgtata catatgtaac taacctgcac attgtgcaca tgtaccctaa aacttaaagt 62700 ataataataa taaataaata aataaataaa taaataaata aataaaaaga attgagtttt 62760 gggaacttcc gcctagattt cagaggatgt atggaaatgc ctggatatcc aggcagaggt 62820 gtgctgcagg ggcagagtcc tcatggagaa cctctgctag ggcagtgcag aagggaaatg 62880 aggggttcca gctcccacag agtccccact ggagtattgc ctagtggagt tgtgagaaga 62940 gggccaaaat tctccagacc cctgaatggt agatccactg acagcttgca ctgtgtacct 63000 gaaaaagctg cagacactca atgccagcct gtaaaagcag ccaggaaggg gacagcccct 63060 gcaaagccac aggggcagag gtgccccaag accatgggaa ccggcctctt gcatcagtac 63120 aacgtgactg tgagacatga gtcaaaggag atcattttgg agctttacaa tttgactgcc 63180 ttgctggatt ttggacttgc atggggcctg tagccccttc attttgacca atttctccca 63240 tggaatagtt gtattaactg aatgcctgta cccctattgt atctaggaag taactagttt 63300 gtttttgatc ttacaggctc ataggtgaaa ggaacttgcc ttgtctcaga ctgagacttt 63360 ggactgtgga cttttgagtt aatcctgaaa tgagttaaga ctttggggga ctgttgggaa 63420 ggtgtgattg atttggaaat gtgaggacat gagatttagg aggggagagg ggcagaataa 63480 tatgatttgg ctgtgtcccc acccaaatct catcttgaat tgtagctccc aaatgtcatg 63540 atgatagaga caggaggcag ccaaaggttc cgctaccctt cataacccac ctaccccagc 63600 cccctccacc cctgtgaaac cctaccttca agtctaaaac agcctgaagg ctgaaaaacc 63660 agactgccaa tctggatgaa gcccgccctt tcctgattga ctctgaataa tgcccagctg 63720 tgcactggaa gaatgggacg tagcctcagg aaatgcatgt catttgtggg gggtggagcc 63780 tggcttctcc tgttcctggg tggggacctg ggattcagtt tgtgaggtgg gaaacctgct 63840 cacaggactc tttcctgctt tgctgagagt tagttttcct ttttgcctaa taaaataaat 63900 tttgttcccc ttcaaccttt aacgtgtctg tgtgcctaac ttttcctggt tgtgtgacaa 63960 gaacccagtt tttttctaca acatttttgg tggccaacat tcggcttgag gaagggtgag 64020 taccgtgcaa accaacacat cttttttcct tttgcttcta agcctttttc tcctcagacc 64080 tcttctgagg ggagaggaaa ttgtgcacta ccccaccctg atggctgcag gcatacacag 64140 gatgggcaaa taaatggtgg gttcctcgct cccctccctg ccagggctgg gctgcatggc 64200 ccaagggtgc ccaacagcag gctggtcaat gctccctgcc atgcaaccat ggagccttcc 64260 cctcccccgg ccaaaaattg tactctgatt gactgcaatt aaatatatct cccttgtgga 64320 ggaaactatt tgcataagaa taagaggttc ttccacaggt atcttttctt ttctccaccc 64380 tgtcaacagg taacacagcc ctgcatttaa acgtcttttc cttttctcca ctgggtcagc 64440 agttgactta agcaagggtt tttgtttttt gttttttttc cttttggaag atgttttgct 64500 aggccaggag tgatggggat cattgtttat attttctgta gagttttaat tgtgacaaat 64560 tctttatgag gttggtttta agctgtagcc aatctggtat gctttgcatg attttctttt 64620 ttttttttaa agatggagtg tcactctgtc acccaggctg gagtgcagtg gcaccatctc 64680 agctcagtgc aacttccact tctctctctc aagtgatcat cctgccttac cctctcaaat 64740 agctgtgatt acaggcatgc atgcaccacc acacccagct aatttttgta tttttagtag 64800 agaccggatt tcaccatgtt ggccaggctg gtcgcaaact cccgacctca ggtggttcac 64860 ctgccttggc ctcccaaagt gctgggatta taggtatgag ccacaactcc aggcctttgc 64920 atgactttct gtatggtcag cagtgaactt tgctgcaggc ctccatcttg gtttatggac 64980 ttgggggcat gacgtgtaac tccatggcaa tgttttgctt agcctctgca caacccaggt 65040 tcagtcatgg cttagcaact gagtcctttc aggtttgata tctgtgtaac ttttctattt 65100 gttgattctc ttctcctcca tgaaccatct tggattttcc tttctctgag gctttagtaa 65160 agtttgaaag gctgaaatac tggcttcttg gtatggctaa agtcaggtaa taggagattt 65220 aaaaggattt tcttaaggag tgctcaacta aattaaagat gaatatctaa gttacaggta 65280 tatttaaaag gcctttttgt tttattttat ttttttctct tctgggatct tgctttgctg 65340 gaaaacggct ttttctcagt tggctgtatt atttttctcc attctgcttt gccaatttta 65400 atgcacacaa gagaggggag agatctctgt cttcctcatt gaaccccagg aattaaaagt 65460 ggatagatcc ctctcaaaat ctcttttcgc ctcccagtaa tgcctgccta ttaggctcta 65520 aaagctgctt gttttcctag ccctccctct taaagggacc aataatccaa atagaagatc 65580 agaaaatgaa aaatcgtatg gctactgggt tttcttcttc ctgtctgtgt agttatatat 65640 gtgttgggtg tgtaatgtct atttaaaaaa aaggtctaat taattggcct aaaagaagat 65700 aagtgcttgg atcaaatatt ttttaaaggg taaaataaaa gctgtggtac ctttcagttc 65760 atatgacttt catcttcaaa aaatttaaac agcctaaaag attattggta aagtgcagat 65820 gtcatcaaaa tataaatagg tggactaaat tatgcaggtc aactgctagg tttgctaaat 65880 gttttaaggt cataaactgc tttttgggtt ttgagaacta tttgtcttgc ctgctccaca 65940 attggtaagg cctggggaca tatagaaata accacgccct taattatgct ggaattagtc 66000 aaaccttgga tgcacctagc acataatcaa aacaacttac caagttttac attaaagtta 66060 aaaattgcta ggagtaacca ttataacatg taattgaaac tactggaaat agatttacat 66120 gcaagctgta taagaacagt aggatgtgtt tttagtaaaa gattataaga aggtgtggaa 66180 atgtaagttc ttgcttaggt ttaaaagatt gttttgaatt tgataagata aagctaaaag 66240 tccaaacaag ttgtaaagga attgtacaaa ttaatcttgc aaaaattcaa tatgtgaaca 66300 tattgactaa attcaaaagg atattatatg gttttcttgt aaattgagca ttaaagtaaa 66360 agcaaaacaa ggttctctta aggcactaat ctgctcttta gcaaaatttt taaggggtta 66420 taaaaagttt tttgtttttt aaatttctga ttcatcattt tggcaaaata aataacaagg 66480 taatctggag ttctatttca taatatcaag tgttttaaac cgctaacaca tttaacaggc 66540 ttctgaaaat caaattcagt ttcaaaattt tctttcctga tgcctggctt tttgatgctt 66600 cagagagccc ttggagtatc caaaagagag gaaaacagga ttatttgaca tatttaggta 66660 tatgagatta ccaaaatggt gttcaatatt ctttaggtta tattttggta aataatacta 66720 atatgtgttt caaatttgta tgggatttta aaaattctaa tgtctgagta tacgctatca 66780 taattaaggt ttttatgtta aataattgta aatgacagag ataaccaagc ttctttgtca 66840 attgtgtttc taactgtaac taacctggac attttgttat tcacagacaa ttttcttgtt 66900 ttaattcttt tcaaaagatg gcttataata agctgtagaa ctctgacagg tgctctcaaa 66960 tacaggtttc tgataacttt ggaataaagg gaaaacacac agaactcatg aagagctaaa 67020 atattcacaa acccataaaa aaactgaggc aatctttttg acttttgctt ggaatattgc 67080 tgatccttgt ttttttttca gagtcaagga aacttatttt gaactattta tgacttttaa 67140 taattgagta aggtatactc ctgtcaacaa aatacagagc atgtttgtct ctctgcctgg 67200 cttctccaga atttgcaaac tagttgtgaa tattcttaac ttatggcaat ataaatgttt 67260 gcatcagtgc aataagaatc cattttcttt tgcaagaggg tgcaattgat aaactagttt 67320 ttttaccaag gtgttgactg gaagggtatg cttcctttta aggagtcaag ctcaacttgc 67380 agagccgata agagtttctt gagaaaactg gcctcatacc cttgtctaca cagtccctct 67440 acagggtttc tgacctgtgg ttagtaaaga atgtcacttt cttacaggct caggagctcc 67500 aagtttatct tgggacctta agaggagacg attacccaac tcacaggtat ttgagcatat 67560 aaactgatgg ctgggcttgg ctttaaaaag tcttatctga gattcctcgt ggaacagatt 67620 tccatcaaaa ccaatgtaaa aggcctatgt agaaatagtt attccttctg cactttatgc 67680 aaacactcag cccaagtgta agattaaagt ctattttaca aacaactcat ccctatcatg 67740 atttttttta aacaaaattg aggattggag agagagaaat tatgtttcaa aacttatcac 67800 acatttgtta ttaaattcta gactcatcag ttgtttttaa gtttttgcct acattttaga 67860 gtaaccctgc ttgttcctgt gaaccaacca gtaatctcca actaaagctc agaaggagta 67920 aaagggatgg gtaatgtcaa aattttggat caacattcta gttctgagca attagcctgc 67980 aaatcctgcc aggtgatggg aataaatagg atgcccatca cctggaggtt tcctttttgg 68040 gaaagtaaga ccaagggagc taaccaaagc caagccccat gcacccaaat cttagcaaag 68100 ataactatag ccaccagtta tctgggcatg tcacaagacg ccctcttcct tgttggagga 68160 ggactcaatt ccacagcctc acctaagcat ttggcttata ataagaaatc catgctagcc 68220 tctgagacac atttttgtcc caaactcaat tctaagcttc acatcaaagc cctgggggca 68280 gggggaactg gatctgaagg acccagatgc agatgataat gcaagttaaa aggcacaatg 68340 cagatgagtg tgactgattc ctgctgaata agccaagctt cccatttcat gaataaaggt 68400 cacactagta tccatggcat aaatgaggtc tggagaatcc aaaggctatg gacagcaggg 68460 gagatagggt atacaagggt aaaagcgaat actctcaccc ccagaccccc ctgttaacac 68520 aagtgaagac cactttgaca cccaccctgt cacagtatct gggacttggg gatacaagga 68580 aggaggaatc tgctcccctt tttgtagatg agtagccatt catcattagt ctgtatacct 68640 ttctttcttt tttttttttt tttttttttt gagacagagt ttcactcttg ttgcccaggc 68700 tagagtgcag cagcgtgatc ttgcctcact acaacctcca cctcctgggt tccagcaatt 68760 ctcttgcctc agcctctcga gtagctggga ttacaggcat gcaccaccat gcttggctaa 68820 tgtttgtatt tttagtagag acagggtttc accatgttgg ccaggctggt ctcaaactcc 68880 tgaccttagg tgatctgctt gcctcgacct ctcaaagtgc tgagattaca ggcatgagcc 68940 actgcacccg gccatctgta cccctttcaa atgcatcctg agtttctagg acccctttga 69000 aaaaaaagac ccttcttttt tcgtgtttct cctctgtcct ctcttcacag ataggtaatt 69060 gtgtttccgt actatgggac acctcaccca gatgcattct ccaaactggg gagagttaat 69120 ttctcaaact ttaacctagt ttgcttagga ttgggctcag gggaagggaa cccagaagcc 69180 tgacatgctg gctaaagggt aaaagttttt tttttttttt ttaccagtta ggtttttggc 69240 ctccctctcc ctgtgcaaac tggtaaaagg cctcagaatt ttttagctgt cctcaacccc 69300 acccccattt tgttttgata catgttttct ataacctggt ttatttctca ccttcaggca 69360 atcaaactcc aaacgttcat gcaactggag acttggatga gggccccttt tgccagggac 69420 cctttgatag gcttctgagg gagctctgac tgccgttttc cccaagcagt gccccctgtc 69480 agcagaaagc agttcagatg agtctttgtc cttatcctta ttccaacagc agttaggtat 69540 acttctttag agggggaaat gatagagaca ggaggcagcc aagggtcccc cagtgaaacc 69600 ctgccttcaa gcctaagata gcctgaaggc tgaagaacca cactgctggt ccgggatgaa 69660 gcctgccctt tcctgactga ttctttctga atagtgccca cctgtgcact gggaggatgg 69720 gatggaacct tggaagtgca tgttgtttgc agtggggagg agcctggcct ctcctgttcc 69780 tgggtgggaa cttgggattc aatctgtgag atgggagacc tgcaaacagg actctatctt 69840 gctttggtga gaattagttt tccttttcat ccaataaatt ccattccccc tcacccttca 69900 aagtgtctgt gtgcttaacg ttttctggtc ctgtgacaag aacccggttt tttgtttttt 69960 gtttctaaaa caatgggtgg aacctgacgg gaggtaattg aatcatgggg gtgggtcttt 70020 cccatgctgt tctcatgaca gtgaataagt ctcacaagac ctgacgattt tataaaggag 70080 agttcccctg cacacgtctt cttgcctgct ctcatgtaag atggtacttt gtcctcattc 70140 atcttctgcc atgattatga ggcctcccca ggcatgtgga actgtgcatc aattaaacct 70200 ctttccttta taaattacct agtatcaggt atgtctttat tagcagtgtg agaacagact 70260 aatacaatga gtaaactcat tttaattagt ctttctagaa aaggtgccaa tctgaagaat 70320 ttcttaaatg agtaaccaca gcataggtta taatgcataa aatattgaaa taatccatga 70380 gtccacaaat acatttaaaa acatttaaaa attaggaaca tcagaagatg acgttctttt 70440 atacaaatga gtgccaacaa ataaatatag aaagaatgct agaaactgaa aagtcatcat 70500 cttgaaacat cataaaagta attatttcag acaggattca ttgatttatg ctaataacat 70560 tgtgtgaaag atcgggaaca aaatactcta acctaaaata actgcctcta cagattactt 70620 gttagttaca aaaagaaaaa ggtacattta caaagtagaa attttgcaga catctcctta 70680 atcaagagat caaaattact gtcaccaaat ataggagaac ctgagatcac atgcttcctg 70740 atatgtacac gcactgtgaa ggatacaata tcacctatgc attattccta ttgtaggtat 70800 ttgacctgaa cttaatcatg aagaagaacc caactcttaa ctgagaggca ttttgcaaag 70860 cctctgagat ggatactttg aaaatagcaa tgtaatgata agacaaaaaa ataaaaaaat 70920 taattaatgt ctagggaaat tttctggatt aagggagact aatagacatg caaactaaat 70980 gtcagctgtg attcttgatt gtgttctgga ttaggaaaat taaacatcat aaatttaatt 71040 tgaggaagaa ttttgaaaat ttgaatatga attgcatatt acataaagtg ctgtataaaa 71100 gttaagtttt ctaaatgaga tcactatatt gtatttatgc aggaggatat cctgcttaag 71160 agatgggaag ttcatacatt taataatgaa gatacagcaa atgtgggaaa atgttaaaaa 71220 tcattgaatt aaagtaaagt atatgtgggt attccttgta caatttttgt aactttgcca 71280 taagtttgta ctttttaaaa taaaatgttt gaaaaattac aataattttc atcataatat 71340 gcaataaaaa ggacaaactt ctttttttga aaaaattttt tttgaggctg aatcttgctc 71400 tgtcacctag tctggagtgc agtggtctga ttccagctta ctgtaacctc tgcctcctgg 71460 gttcaagtga ttctcatgcc tcagcctcct gagtagctgg gattacaggt gtgcaccaca 71520 aggctgagct aatttttgta tttttagtag agataaggtt tccccatgtt ttccaggctg 71580 gtctcgaact cctcacctca cgtgatctcc ccactttggc ctcccaaagt gctcagatta 71640 caagtgtgag ctactacgcc cagcccaggg acggacttac tcactcattg agcatcattt 71700 ttctttgcag ttggatagct aaacaaacaa aacaggcaaa aagtccttcc tttgtagaac 71760 ttccattctt tctccttatt tctgagcttc cattgttaaa tagatgaggg tggggaatga 71820 taataagtac tggatttgat attttgtagt ctctttcctc actacatgga attatttcag 71880 atgcatgaat ttgtttgctt tgaatagata taccgtggct aatttcttcc acctagagct 71940 ctataaaaat attgtaataa aaacactaac caacaacact tatgcatagt ccataacaga 72000 aatggcccta ttagttctgt cttttgtatt attacagacc caaagcaaga caccactgaa 72060 ctatttatta ctttaaaaac attctaagct gtgaggttgc tttggttttt ccttctgaga 72120 ttaagaataa aatatctatt tcagattttt aaaaaagcat tccttagatt gtctttgttt 72180 gcaggccttg taggcactat cttgtttgtc taaatctaaa aagattaatt atatcattat 72240 taatggtcat gtatcaccaa cagaaaataa aatataaaat tttaatttaa agctaaatta 72300 aaaagtatta gtcaaagatt ttttatacct ttagatatat gccaatttaa cagaatcaaa 72360 atttgtatta aaacacataa gactttttaa aaatagaatt aacaggaaat atttgactaa 72420 cacatattta tgtgtctata taactggtaa tgggtcagaa agaatatact cctctagaca 72480 ctgcatattg tttttaatgt tgcttaggct ctaatttcaa atgcagggtc agataaaagt 72540 tggttgcatt gtttttctat gcatgctttt taatttcatc ctttgcattt attaacacta 72600 ataggtagtt tttttttctt gttattgttg atgctacctg ttcagaggta ttggataata 72660 attattcaga actctagatt ttcactgggt tttgttttgt catgtaatta cctaccttat 72720 gtgtgtaggt atttgggtgg tatgggagag aggaagaaaa tttgaaatct atctttttaa 72780 atttttttct tatttttttt tacaagtccc tctcaatgaa aatctacctt tgtcaaacac 72840 atttgctatt caaaacaact ttaatgacca gatgaagtag aaggaaggaa agtgggcact 72900 tccatatttg tttttaatga gatatcttag aaagttaagg aacttgaggt cacaaaggag 72960 aagcaaggta taactgtgaa gaggatactg gctttgattt caattagggg agatgaagga 73020 aaatggaagg agatttccta actcaacaga gaagatggaa tttaaattta ccaaaatcag 73080 taataaggta gaagtgagaa tgaagtactt tcaaagctaa agacagaagg agaaaagtgt 73140 ttaaaagcta attttaaagt aaaagagaga tgaggagtta ccagatttgt gtgagaaaga 73200 gcaaatacat ttactgtttt taaatatcat agaagagagg aacaaatttg agtaaataag 73260 tttaagaaga agcttgcttt ccccttatta tttgttaatc agcaagatag tggggaaaat 73320 gtcttcagga catgtcagag acctttatgg cagcccctcc catcacaggc ccagaggcct 73380 aggagaaaaa aatggttttg tgggccagga ccagggtccc cctgctatga gcagcttagg 73440 atcttggtgt cctgtttccc agctgttcca gccatggcta aaagggccaa ggtacaactc 73500 aggccatggc ttcagagggt gcaagcccaa agccttggca gcttccatgt gatgttgagc 73560 atgtaggtgc acagaagtca agaatttaga tttgggaacc ttcacctaga tttcagatga 73620 tatatggaaa cacctggatg ttcaggcaga agagtgctac aggggcaggg ccctcatgga 73680 gaacctctgc tagggcagtg caggagggaa atgtggggtt ggagccccca caaagagtga 73740 gaagagggct gtgagaagag ggccactgtt ctccagaccc cagaatgtta gctccatcaa 73800 tagcttgcac catgcacctg gaaaagctcc agacactcaa tgccaaccca tgaaagcatc 73860 caggaggggg gctataccct gcaaagccac agggtttgga gctgcccaag gccatgggag 73920 cccacctctt gcatcagcat gacctggatg tgagacatgg agtcaaagga gatcattttg 73980 gaactttaaa gcttaatgac tgccctattg gattttagac ttgcatgggc cccatagccc 74040 ttttatttgt gccaatttct ctcatttgga aatgagtgta tttacctaat acctgtactc 74100 cgttgtatct aggaagtaac taacttgctt ttgattttac acactcatag acagaaggga 74160 cttgccttgt ctcaggtaag tctttggact acaaaatttt gagttaatgc tgaaatgggt 74220 taagacttgg ggcacttttg ggaagacatg attggctttg aacaatgtga agacatgaga 74280 tttggcaggg gctaggggca gaatgaaatg atttgactgt gtccccaccc aaatctcatc 74340 tttaattata gcttccataa ttcccatgtt ctgtgggagg gaccctgtga gagttaattg 74400 aatcatggag gcgggtcttt cccatgctgt tctcatgaga gtgaaacagt ctcaagagat 74460 ctgatagttt tatgaaggga agttccccta cacaaactct cttgcctgcc tccatgttag 74520 atgtaacttt gctcctcttt caccttccac catgattgcg tggtcttccc agctatgtga 74580 aactgtgagt caattaaacc tctttctaaa ttacccagtc ttgaatatgt ctttatcagc 74640 agtgtgagaa cagaataata cataatttaa ccctaaagaa gagctcttta atggagaaat 74700 tcatatatca tgaaatttat cctattcctg aaatctttac tgtgataact gaggacctct 74760 agccagattc cttacttaca tcacttggaa ttagtttgcc tccgtataag actagtttga 74820 acttatagtg ctgagtttac aagtttccaa atatatgcaa aatatacgta atcatactga 74880 tgattaatgg tgagatgctt ataaaatggt gagtctaaca agctctatta ggtacagata 74940 catgcaatca ctgaattctg tcaagagatg atgcccttta tgcacaataa tgcacattct 75000 tccatttatt attcaatata ataaattcta acaattttcc aaaagtgttc atataaaaaa 75060 taatcagact ttatgtttta gtaagttgtt tgttttcagt aatcatttat tcttgagcag 75120 atattttttg gtgttttatt ttgtcatgtt tattggctat tatttctgtt agaaacagct 75180 tctgttacta agaaagaaac accgacaagg gaagacacct ttcccataaa aaaaaattta 75240 ttccagctgc gttttgattt gaagttgaat aaagacaaag gtaaaaagaa aagctaagaa 75300 gacgttcccg ggtgagtcat tggagtgtca actagcctgg tcaggggctg ctgcttaact 75360 acacatattt gataggaagt gtccctttaa ctgtgagaaa atggtttcct aatgatactt 75420 tttgcttttc actcatgaaa tcactctggg ttagcttctc atcagtccat aaatgcccat 75480 acctggttct gtcaacattt ttggttttca gattatgtgc tttatgaggc aaaacatagc 75540 aacacaaaca tttaaattgc ttccatgttt tacaacctca actgctgtat tttatataaa 75600 gggcacaaaa tgaatactgt tattgatgaa aattgggtta ccctgaggac tcttgcttaa 75660 ctgaaatcac aaatggagca gacagaaaaa tcaaaagtat atgctgagaa cggtgagtaa 75720 agagtgaact cctcagagca gtaatatttt tcatttctag aggccagaag taagaaaaga 75780 gagatttttt ttcttcattt gggaaatttg atttccaatg ttacttgaac acaatataag 75840 taatataagt tttaaaaatg aatagtttta cttagaaaaa aagttatttg tatgagggac 75900 tcaaaaatag ttttaaaata ttactatatg ccagaatctg attttgtttc tttgttttgt 75960 tttatttctt ttggtttcta tttatcctgg gcctgcaggt tttatttttt aaatgtaaca 76020 aagctgattt gatgttcctc aaaatgcttc tatgttgatc agcactcaat gcaaggtcta 76080 gttgtcataa tgacactgta tatactccag tcaggagagt aagaagtttc tgctttgcaa 76140 actagtgggc catttattca accaatttta atcttcctga cagtcataca tttacctaaa 76200 acaataataa aaaaaactct attaaaacac tagaaaatag ttggtattac tccagaaagg 76260 ccaaaccaat tgtcaaggat tagatgtata ttaaatgggc tgatttttga caggcaaact 76320 tgaaagttaa atgttttggg gtaatgcttg caactagcca cactttgctt gtcagtaact 76380 agttactaca ttagtcttcc ccttctaata gttttgatcc ctattctagg aatggtacaa 76440 gttatccaga gactgagttg ttcttttatc agagtaaatt cttcgcatat gtttgcaagt 76500 catgactaaa gaatacacaa aagagcatat ttaaatatat tttgctaatt agtaacaata 76560 acttttttct tgtttctgat ttttctctgg gattatgcca gttacttgat acatgttcat 76620 taattttata ctaaaaacaa cattaataac ggtgctattc taagatttta gtccaacttc 76680 aagctaatca tattaataca agctatcctg cttagtgaat aataatcata atataatttg 76740 taaaataata ataatataat ttgtaagtga atgaaatgga ctcccttaga aaatgttaat 76800 agacaccttt ttatagcata tgtcctataa ctgaataaaa agcttagcta attgattggt 76860 tatttatgtt gtattaaata atgatttaat agtaaatgaa gcttgaaaca tcttgtaagt 76920 aaattaatgc tggatagttg aatacctata ttccaaaagt attattttgg caaaggatat 76980 aaaagttaat tcacctaatt atgaacatat ataaagccat taagaactaa ttttttaaga 77040 tacttttaaa gctttttaaa attcctgaat attttactat ttaccacatc accactacat 77100 tagcagaata ataaaaatag ctaataccct tgagtactta ttatgatcta gataatgtgc 77160 taagctctct ccaggtatta tcaacaaatt taatacaaca aattcataag gcagatacca 77220 ttgctgccat ttgacagaaa aggtataaga ctaagaaact tggctaaagt tgtgcaggtt 77280 ataagaggcg aggaatggac cggaagcctg atcttccatt gtgaagccca tctcatgctc 77340 ttaactatat tgaacctttc cctccacaaa gggattcatt gcctcttctc ctatgaacaa 77400 atggaaagaa atttaaccaa aataaaattg tcaacatatc atatattgcc aaaaaaattt 77460 agtgttacct tccctggttc ttaccaatga atatcaataa atattattga ttgattcctg 77520 atagacttga aatgatgaat taattccatg aagaggtaga aaatagatta cttaactaat 77580 actacaactg aacactgaac attctgacta aacaaccaac aaaatataaa atagttgtac 77640 attaaagaat acgagaatga aagttacatt taccataaat tataccacca taggacaaac 77700 attagttaag aacagattac ttttagccat acttaggact gtcagaggaa tcagcctcat 77760 tttgttcatg acttctaaag gactttcaga gagaaagagc attttaaaaa taagagttaa 77820 tatttaaagt ttatgaattg ttgtaaccaa cttggtttta aagctggaag tcacctatta 77880 ataccatacc actaacacat ttacattaat aatagccgtt ctatgaatag cattagcatc 77940 ttctcaataa acccatgtag ccaacactta cacttcttga ttcctgaata aatcatgcaa 78000 caaaagatga atttttagtt tctatgaatt acaagtaaaa agagtcacct ctttaaatta 78060 tgtgagttgt gtttttttac ggatagcatt aaggtttcct aattactttt ttttgtttaa 78120 tcattttatg ggcaaaacct taaggactct tagaaaagat aaagctttgc ctttcaaaga 78180 aaccactgcc atctcccact gagcgaaaga agtttgacca tgactttgcc atctccactt 78240 cctttcatgg gatacacaat attgttcaga accggagcaa aattcgcagg gtgctctggt 78300 tggtggtggt tctgggctca gtctcacttg tgacatggca gatctacatt cgcttgctca 78360 actacttcac atggccaacc acaacgtcca ttgaggttca atatgtggaa aagatggagt 78420 tcccagctgt gacattttgt aatttgaaca ggtaaaaatt acttttttaa aaataattag 78480 ttcctataaa tttgctagta taatctttct tggatgactg acaaagagtt ttctcatgtg 78540 gaacttatca tatgtaaaac cactgaatta aattgcactt aaattgcact caaaagcaca 78600 aatttaaggg agcaaagaat tcattaattt cttctttcac ttattttcca tcttttcttc 78660 cttccttcca caatcatatt ctaggtaaca tcccaaatcc tatcattaaa tagatgtaaa 78720 agagatgggg gtttcactac attgcccaga ctattctcaa actcctaggc tgaagcgatc 78780 cacctgcctc agcctcccaa agtgctgaaa ttatagatgt gagccactgt gcccagccag 78840 atgtaaagtc ttgatccctg catattagaa gttcattaat tagcagaaaa gagaaacatc 78900 aacaatcata gtgcaatatg gtaattgcac aacaaaataa aacaatgtgc aagggctctg 78960 aaatcccaca gaacagatta atagtctcca tcttgggtgt tatggaaatc tgggtattaa 79020 aagatgatta cagctttcta agtggagatg aagaagaaat ggaagaggga actactttgc 79080 aaacacataa cgatataaaa tgttctggtg tacatatgat gagctactta atgtagtaga 79140 ggcaaggcca gctccacggg tctgtgatca gtgcggctgc aaagagccct gagcttagaa 79200 aagggttaca cttgattcaa tgctctactg ccagtgtctt gagtccttga ctgcaactca 79260 ttccagaaac ctgcagtcca ttgactgtac accaaatctg ctaagaggag gacaactttc 79320 caccactagg cttccagaca atgctttgta attgcctgtg gtgaagcatg aaataccata 79380 cataggattt ttgctgagga agggagggct tcttaagact caccacttag ctattaggga 79440 agtcactcag ccttcttagt gttaattttc atatttataa aatgagataa tatcatctac 79500 ttcacatatt tgttgttaca ttagataagg taataagtac tccattgtgt ttcattcctg 79560 ggggacttct agaacaggtg ttcaatatat aatagatgtt atagcccgtt tgtatgattg 79620 tcaatatttc aagaactcaa gatatgtatc aagggtccta acagtcgaaa caaatcaatc 79680 ctttcccccc aaaaaatgat tttcaatact attgcttcat actgttttaa gtagcatgtg 79740 ttctaacaaa aacagagatt ggttgcagtt atccttgact gaaataaaat gtagatatta 79800 cttccagaat ggcaaagtga ggaagtccat aaactctatc cccttcaaaa ccaaccataa 79860 ctggtgaaaa aatatttatc ttaaaaggca aacttttctt ttaaaaaaga aacatttaaa 79920 tagtatctcc tcaaattcaa gtctactgga acctcagaat gatcatattt taaaattggg 79980 tcattacaaa tataattaca taagttgagg ttatactgca ctaggatgag ccttaaatcc 80040 aatgactgaa gttcttataa ggagaagaga gaacacacag agatacagca gggaaaattc 80100 atgtgaaata gaagcagaaa ttgataaggt tttgccacaa gccaaggtat acttaggaca 80160 aaaggcataa gacatagaga aaacaaaaag taaaacggca gatgcaaatc taactatatt 80220 actaatgaaa ttaaattaga atggattaga caatccaatt gaaaggcaaa ggttatcaga 80280 atggattttt ttagaaagat ccagctatat gttgtgtgca ggagacacaa tttagatttg 80340 aagatgcaaa tgggttaaaa gtaaaagaat ggaaaaaggt atgtcaacct tcagaaagct 80400 agagtagata tcctaatctc agacaacata gactttaaag taatatatgt tacctgatat 80460 aaaaaattgt cattttatgt tgaataaatg atcaatctat caggagatat aagttaacct 80520 atatagttag atttttcaat accccacttt tagtaatcaa tagaacaact tgacagagaa 80580 taaacaagga aatagaagac acagaagact taaacatctt tataaaccta ctagactttg 80640 caaacatcct tagaacactc tactcaacaa tagcaaaata tgcattcttc tcatgtgcac 80700 atggcacatt ctcttagaat ggaccatata caaggccata tannnnnnnn nnnnnnnnnn 80760 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 80820 nnnnnnnnnn nnnnnnnnnn nnccccacct gaaccactca ggtttataat aggtagagca 80880 tgtattgctc tcaactcagg tgtatttcat ctttacctgt agctaaagga ttctagctct 80940 gattcaccag gctcagtaat tgatggacac aatccatgga gcactatcag ggttttcttg 81000 atttgggagc acgaagaggg gacttcagac tctccccatg caggctcaga tggcttgcca 81060 cattgattct tcctttactg tcacttaaac tcaggattac attgctccca ggcttcaaga 81120 tgggcctagt ctagacaaaa actttcagtc tcatgattat gtgaaactct caaataatgt 81180 gtaccatgaa gtgtagaaca ttgcctgaca ctgagttagc tctcagtaaa ttacttgtag 81240 caggaattgt actgataatg caaatcattc caatcttctt ttagcttttc ttgcagtgag 81300 gttggctgtg tcacttgttc aaggaaagag aatgtgaaca gaaggaatac gtgtgtcctc 81360 caggactgaa cttaaaaatt tgccacacat actccccaag attccttctc caactgccca 81420 gtagatatat atactcaagg tgacattgaa gatgagagtg atctgttggt ccaggtcatg 81480 agtggctggt ggagcttcag ccctcatcac cctcatcctt agataatatg tgagaaagga 81540 actgctcctg tgagtcactc aaattttgag tttgtttatt gaaagattag gctactttca 81600 aagacacagt accagctcag gaccttgcag gctgaagatt gcttactgtg atgataactc 81660 tgcttccatg aacatccgtc ttagggcaag aagctgagta aattatttat cccacactct 81720 atccatccaa acaggtgagg ggctgtgcct tgcccttgtg tgaagaaacc aggttctatt 81780 caagagtgtt actctttctg tgaacaggag gcaaaggtat gagctgagac tctacctgct 81840 ctcaaaatca tcagaaagag ggctcagaag gagtcacaat tactgaactg ctgactgaaa 81900 gactctctgg ggactcaacc catggctatt aatgtcagtg tcactgggcc agctcagatg 81960 caaacccacc actactgtgt catccacaaa atacatacag ctatcgcata tcagcattgt 82020 gggagaacgg tattaaagtg aacttcactt tatagaaact gtattcctga cagccacata 82080 cagttcttat gaccatgaat caaatcgcat ttatatgaac aaaggcaagt aatttaatag 82140 aaccctatta cagaacatag gattctctac aggacagact ttacacagtg aacattcaca 82200 cttagtttta tcattaattt tcatagggca ggtttgctgt actaactagg catgcaaaga 82260 ccatgggtct cagcaggtaa tataaagtga tgaggcaggt tgggtggatt tgaggtaaaa 82320 gtgagcgttg gggaatgcag tagatcatga atatttaccc cacccaaatg aggcagctcc 82380 cactcaggga caacggttca ttgtagagac attgtgggac aagtgatgct gcatacaatg 82440 agtgatctcg atttctttgt gaaatcatat gatttaaaaa tgttagcatg ataacctgat 82500 cctacttgga agttaataag ttagcctgct gcattttgta atgctggcaa aaaccgtaag 82560 atttctgggt cagtgagtaa agaatgtatt actcacagca acagcaatag ccagagtagc 82620 cagtttcttg agtcccaagt ccacagggtg atgaaaagag gtccaagtaa cgcctgtcca 82680 tgcagtaggc tacatcacaa gagaggaacc ctgaacttag ggaacatgaa tcttttatac 82740 tgaacagtaa gcgtgcctgc cccttgctcc agaaagaaat actatctcta tcttccaaga 82800 cgatttgctg tacaaacacc ccaggaaagc tagtgcagaa caaagacact cagaacctct 82860 gctcataaga catgcataaa tatgagagac cacctcccaa cagtattctc ccctagtttc 82920 tacaccatct tgtcttctgg agaattttcc cttggataca tcagtccatc agcactctga 82980 ttaatctgat caggagagct gggcctaaat ctgttcaatt tggcttatat agcatttaac 83040 tgcagttact atcaacagga cttcaagcag tagaatgagg ccaacctgca aaactgacct 83100 gtcatgccct gcctcaccat cttatccagt cctggactca accagctgaa ctaatcccta 83160 aaccaccaag tctaccttag aaaaacaggt atgtttttcc tcaagtttct ctagcgatct 83220 ttccacttag ccaaaggcat tattgcagat acagcagggt gtatttgcaa ttacacagac 83280 tccaccttgg cctgcaaggg gaaagtctag ggtgatttta ttatcattca taaggaccct 83340 ggccagcaag ttgtgtatta gctgaatgcc tttggcagac aaggtggtat atttaatccc 83400 ttcagctaaa agggaaaatt tttaactgtg tgattcccat cacagggata acagcttgca 83460 ttttgtgcat aaatgattaa ttagttattc ctcaaggaag cttctctaga gttcaaggat 83520 ggcttccctt tgaaaacatc tcatagtctc atgcaggtat catgatctac tgctgggatt 83580 tccttgaaga cacttgaaga tctcttatcc ttcatatgat ggttactttt atgtgtcaac 83640 ttgacttggc atcagggtgc ccagattaaa tattatttct gggtgtgttt gtgagggtgt 83700 ttctgaatga gattagcatc aaactgatgg actcagtgga gtgattgccc tcccctgcct 83760 tggtagggat cacctgatcc atttgaaagc ctgaatagaa caaaaggcag aggaaagaag 83820 aattcagtct ttttttctcc ctcgttgcct gagctagacc ctctgtatta gtccattctc 83880 acgttgctat gaagaaatac ctgagactgg gtaatttata aagaaaagag atttaattga 83940 ctcacagttc cacaggacta gggaggcctt gggaaactta cagctgtggc ggaaggcacc 84000 tcttcacagg gcagcaggag agagaatgag tgctcagcaa agggggaaga gccccatata 84060 aaacccttag gtctcatgag aactcactta ctatcatgag aacagtgtga ggtaatctgt 84120 ccccatgatt taattatctt cacctgatcc tacccttgac atgcggggat tattacaatt 84180 caaggtgaga ttttggtagt gacacagagc caaactatat caccatctca tctcattgtt 84240 tcctgccctt ggactgggat ttagataaac tcccctcagt ctcagttctt cagatttgga 84300 ttgaattata ccaccagctt tcttgggact ctggcttgcc ggcagacagc agattgtggg 84360 acttttcagc ttccataatt acctgagcca gtttctcata ataatacata tttttggaaa 84420 actctaatat agattttggt actgagactg cctctaaagg aacatattaa ggatgagttt 84480 tctgaattca ttccaaggtt tatggaactg gctctctaat ctgattagat ttaaagatac 84540 tggactctat ttccagtagt aaagagtgca gggatagtca gtggcatgat ctggcaatag 84600 agatatgcaa aatacctgca ctatatgctt ccagtgagcc actaataaga agtaaggagg 84660 tgggagactt tgtatacggt atttttgaaa gtttctggaa aactaatgca tataacgaca 84720 tagactggtt tgtcctaatg tcattggaaa agttgcacca aaaaagaatg agctcaggga 84780 ttcaaattct cagccgtata aataatcaaa tagcttctat gggtaccctg aaaaagacgc 84840 ttatctcctg tagccacagg actgaaactg ctaaaaatta aacacaaaac ctcattctgc 84900 aactggctaa attacaatga agttgacttt ccagctttgc aggatttcta cactaaagtg 84960 agggcatcaa ttggaaagaa tgggattctg taaattaaag ataaagacat gagcaaagat 85020 cctgatgaag ctggagacat tgagtcccta aattctgatg agtcttcttt gccagtggaa 85080 gaagtcttcc aacccccact gaaagcggcc tccccagccc cagtggaact aattccctaa 85140 cccctggcaa agtggcctcc cagccctcca tgacagtggc atcctcaccc ccagttgtat 85200 tgggctttcc atccttatct gaggggttaa ccctgcatta cctgacaaaa tggtaatagt 85260 ctctctgaag cattgctatg caagacaatg ctgtttcttc tcagggccca tccgaaccat 85320 ccctctttcc ttttagacct ataactagac tctagtttta gcaggcctct gaacgtgagg 85380 tacaacatat gacccatgag gatgtgcatc acactctaaa agaaccactt ttgttttcta 85440 acttatgcag acaaaaatcc agggaatatg gtgagaatgg atatgaagca tgtgggatga 85500 tggtagaatc agtgtacagt tggatcaggc tgaatttatt gacatgggct cgctatgcag 85560 agattctgca tttagtgtcg cagcttaagg agttagaaaa ggctctaaaa gtttctttgg 85620 ttggttgact gaatcatgga ccacaagatg gcccacagag agtgaactgg aaatgtcaga 85680 cattccttgg tttaatgtag aagaagggat tcaaaggctt agggaggttg aaatgttaga 85740 atggatttgt cgtgtaagac ctactcactt acactggtgg tgtccagaag acataccttt 85800 cgccagtact gtgagaaata aatttgtgag agcagcactg gcatctttga agggctccgt 85860 aatttctctt ttctgtaggc caggccttat tttgggatct gcagtcactg aattaagaaa 85920 cctacagaca atgagggtaa taaaatctgg agtgacaggg accaagtggc agaactcaac 85980 caccaagagc aaagtagatg tggtaacctt aatagagagc agagtcaaag catccatcag 86040 aatagtgtga ctcatgtaga tatatataac attggctact taatcatgat gttcttataa 86100 atgaaataga taggaagcct actaaattct tacctgacat atgtaagcaa aaattttcag 86160 gttaagcaaa caaaagttta acttgaatct taaaaacaga gaatcatggg cctcaatcaa 86220 ttcccagatt tgagccagtt tacaaagtca gagccccttg aatgaaggga atgcttggcc 86280 cccttgagga tgtacttagt ataccatcaa aaacatacac tggtaatgtt tctctcattc 86340 ttccccgaag gaacctccag ccttttacca gggtagttgt gcaatgggga aaaggaaatc 86400 atcagacctt ctggggaagg agatattgat attgatttga gagccttgaa agatcactgt 86460 ggtcctttag ttagagtatg tcctaatgga agtcaggtgt tcgatggagt ttagctcagg 86520 tttaaatcac aatgggtcca tttcgtcact gaacccatcc cgtggttatt tccccagttc 86580 cagaatgcat aattggcata tacataatta atagctggag gaagctccac attggttccc 86640 taacctatgg aatgaagacc tggtaagaaa ggccattgga acttcctcta cctaggaaaa 86700 tactaaacca aaagtaatac cacatacctg gagggattag aaaaattagt gccaatataa 86760 agaagaaaga tacagtggat ggtgattctc accatatacc tgttcaactc tcctatttgg 86820 cccgtgcaga agacagacgg ctcttgtaga atgacagtgg attatcttaa gcttaattag 86880 gtcattactt caattccgct gctgtaccag atgtggtttg attgcttgag caaactaaca 86940 catctcctgg taactggtat acagctatta atctgacaaa tgactttttt ctccatacct 87000 gcctataagg cccaccagaa gcagtttgtt ttcagctggc aaggacagca atacaccttc 87060 actctcccac ttcagggtta tatcaactct ccagctgtgt gtcataattt agtttccaga 87120 aatcttgatc acctttccct tccacaaaat gttacactgg tccattactt tgatgacatt 87180 atattgattg aacataatga gccaaaagta gcaaccactc tctacttatt ggtaagacat 87240 ttgtgtatca gtgggtggga aataaattgg actaaaattc agagccgttc tatctcagta 87300 agctttctag gggtccagtg gagtggagcg tgtcaagata ttccttccaa ggtgaaggat 87360 aaattattgc atttggcccc ttctatgacc aagaaagagg cacaaggcct agtgggtcta 87420 tttggattct gcagacaacg tattgggata tgttgctcta gcccatttat ccagcaaccc 87480 aaaaagcttc tagttttgag tggggcctag aacaagaaaa gacactgaaa caggtccagg 87540 ctgctgttca agctgtacta ccacttgggc agcatgatcc agccagtgtc cttacggaac 87600 aggtcctttc cattgtaatt tacatgaata atacagacat aatcaattgc agtttgtttc 87660 cagagttcat gatccccaaa atgggtgcct tcaatctacc aatatgtagt attccaaata 87720 gcagaccaaa cagctaggac attagcaaca acctaagaat aagtaaaaat aggacacagt 87780 tgggtttcag gttagctgaa ccatcaggga aagaggcacg gacacacaag aaaaatcttg 87840 agttgaaggt cccattgagc cagcagcttt gctgtaaatg gcaaagtgat gaataaaggt 87900 ttccccagag tgatagcagc tactttttat gttaagacaa ggtgctgctg gcagtgggat 87960 agctgcattc ttgaatatac tatttccact taccaagcag ggattgtcag gccctctcca 88020 tcttattaat cattgagtct gagtcaaccc acccaaaatg aaaatatcag gctggagagt 88080 ctcaagactt ttatgggtca ggtattttgt tttgaaggag catagtagca agccaaaaag 88140 gctttacatc aaagtgattt aacagctgtg tcaaggaggt gatgagtcca gacctcaaca 88200 aagacttctg ggtgaaggct gacccccttt atcagaggct ccaatcacca agactatcag 88260 acatggaaac ttgtatctca gaaaggtcat gagggttgtg gggccccttt ggtgcttgca 88320 tttatctact tactgctgtt actgatcagg agaattttcc accacttcct ggataaggca 88380 gtgtgagcac ctcaatttct aatgtatatt cagacatgga tatgacaaaa aataatacac 88440 tgtattggcc aaaaagacca caaacagaag gtcacacttg tctcctttcc ctactctaat 88500 ttctgaccaa actctattaa tggaaagttt gtgtgtccca ttctgacaag ggccctgggt 88560 gtcagtatac aacagaggca taaaattatg attccctccc ctccccaaaa tctctgcccc 88620 cagctatact tgggcagttg cttttggcct ttgattccac ttggaggcag ggagagcctg 88680 acctcacctc taatcattta tctccttaaa gtagctgaga tcagagtaga gagagaggga 88740 tgggttaggt gtaagatgta gagagtagct ttccaccaat aagggaggag cacttacttt 88800 tagttatgag cacacagagg cagctcaacc aaatgaactt gcagtgtatt caacctgccc 88860 aacactctaa taggtggcag attgctcata actgacacca tctgtttcag ctttgggggt 88920 cccttgacca agcagccata gcagcattgt attccgactg tgggcatgga tttctaccca 88980 tcacctgaca tgggattgac tggcacaggg ataatttttt aaaaagggat gcaccttaat 89040 ctgagacatt tggtgcctca ttatcaagca aatatactta agttcttgtc caatttaaat 89100 gcaagtgcta taggagtttt ccaaggctac aggtctgacc tcagcaattt atctacactg 89160 tcagtctctc attctccagt gagtcgcttt catcagtgtt gcatacccaa ttcaggggca 89220 ttaaaagccc aaagattagt caacgcctgt atttgtttgt tagggtttcc ttgataatga 89280 accataatgc ctgggtgact taaataacag aaatttattg tttcttagtt ctggaggctg 89340 caagtccaag atcaaggtgt caccatcatt ggttgcttct gggggctgtt gagaaagaac 89400 gtgttccatg cctcccacct agcatctaaa ggtttgctga acatctttag tatttctggg 89460 tatatctacg taacactgac tctctgcctt catctttaca tgatgttttt cctgtgtgtc 89520 tctatgttca aatttctcct ttgtataagg acaccaggta tattgcatta gtggcccacc 89580 ctgctctagt atgacctcat cttaacttac ctcattatat ttgccatgac cctattccac 89640 ataaagacac actctaaggt acttgaggct atgacttctg tggggacagt tgaaaacata 89700 acaatgcctt atgtatgctt tcctgtggaa gtttgtcagc tccagagaaa tctctccaaa 89760 agggacaaaa ctcccattgg agccaggacc tactgaaaaa cactctgaga cctttaactg 89820 tcatctctga aagaatccaa ggttggcatg aaagactgca agaagggctc agccgtaagg 89880 cttcccagct gttgccattc ttcctcaaca agtgttatgc atctattgcc ctcatctctc 89940 aaatgaacta gccaagttct aatcgcttac tcagtttctg ctcatagggg ccatatattt 90000 tcttctttgt ggtcagtgta aatgtacact gtgttagtca aaggatacaa aatttcactt 90060 aggagaaata aatttaggag atctattgtg caatatggtg actatagtta ataacaatgt 90120 acctcatatt ggaaaattgc tacaagagca gattttaaat gttctctcca aaaaaaagtg 90180 tgtgaggcaa tggatatgta aattagcttg acttagcctt tccacaatgt atacatatat 90240 caacagatca tgttgtttat caaaaatatg tacaattttt atttgtccat ttaaaacaat 90300 tacttgacta atttttgttt ttaaaatgga tatatctgta actcttgtct gaagaaggtc 90360 agaaccttcc tctaacccag gatggttata tgttatgacc tgtaagaatt ttccagggct 90420 acaggtctaa cctcaatcat ttatctcatt gacttgaggc ttgaggagta gacttgaggc 90480 tgacacccag tggaggaaga atctgctcac ccagtgagag ttaacaacca agccactgtt 90540 taggccactg cttttgaacc tactttctga tgcttgagat gcaatgtttt ctgaattcat 90600 tctcaatagc aggtgataaa attggggacc atttctcact ctgctgattt ataatttggt 90660 taccacattt gccatcgatt cccatgaact ttttcctcca atcccattaa tcagctcaca 90720 atacccttac tcttctcttc caggaagcca agtttctgtc agcatcccaa cctcatttcc 90780 aaactgtcaa taattgtgaa gggtctgaga ttttactcta cttgcaagtt aaacagctag 90840 actgccacaa tttcatggat gctggcagaa gacgtgactc ctgggtcaga gaaagggaac 90900 ttcactgact cacacaacta tagcacctga ccaagggtat cagcgttatc ttgcactggt 90960 tcactgggtc acacacagca atgtgaaaac aaataaacta cacctgcaca cagtgagttg 91020 cacgccagaa gatacatcct gagtttagga acacaaatct tttataatgg gcagtaagcc 91080 tggcggctct ttgttcttgg gagagacatt atctctgtct tctaaggctg cttgctatac 91140 agacatcttt gaaaagataa tattaaacaa aggcagtagg tacccctgcc ctctataaca 91200 ttcaggagat cgatagaaaa ctgtctccca acagagtgca atggagacac atttatcccc 91260 cagtttctgt tataagccac tacacttgga taagccaagc acagttttat ttctacggtt 91320 ggtgttttct aaaaagtttg actttatata tgatttgaaa aactgagact ttttaatttt 91380 gttttttata aatagaaata aatttttttt taatttaggt tttaaaacaa ttgtgatttt 91440 atgtataata tgagttttgt tggaaatagg agctttagaa ataaatgtat tttgattatg 91500 ttagtacact atttaacaat aactttttga aaagggattt gaatacattg acctgtacag 91560 agcaaatgcc tgacataata gttgttaatc ctggttttcc acactgttac gttggaagtt 91620 ttctctctct ttagtagcaa gacctacatc aaagcaaata tattcgtata acttgggaca 91680 atggtgtttt tccttgatat aaagttcatt tttcttttta aacataactc attttaaatt 91740 ttaagaagat ttttatttaa acacttttat tacaatattt aggtggcaca ataactaaca 91800 agcttctgag acaggaggta acattctcat agactttgca actcagccag aagtaaaact 91860 cgaaataaat atgtcattta aagtaactat gaaggtaata ataaaaggag tgttgttagt 91920 actaagaagg ttttcaatgc agggtccaat agctatattt acatatacag aaaaaatgaa 91980 attagttact aaacataaca aaaaaaactt ggtataccta tagcaatgca tatttaccaa 92040 aaactggtga aaaatatatt gagagatatg ttaaatattt gctgaaaaga aaactacttg 92100 tctgtatgtg aaaccccatg aatcatttta cacatcagga ccaaaaaact aactcctttt 92160 attcctcaaa aatcaaaagt ggtctcatct aacccaaaaa tcacagactt gtcagttatt 92220 gtcttctcat tttgaacatt atttactatt ttttctctct aaacagtaag ttcttagtca 92280 aaatttatta ttttgtccca tctagacaat atggtctatg gtacttcact agggtggtct 92340 aggagtagat tctagggagt tccccaagct ctggcagtac atgtgtcttg tatggggcca 92400 tatgcacatt tttggagggg tgatagtcta tgtcttttat ccatttctca aagcaataaa 92460 agtgaagacc cactagccaa ctgtagcagt ttcccaagta aaatataaag atgattcact 92520 ggggtaggtg aagaaaatac tagaatttct atttatattc atgctcattt agaaaagaaa 92580 agctgtccta atatgtagta tatggattga gagcagcatg tatcagtcta tagagttgtt 92640 gtcctacaat attttattaa cagattgcag gaatctaaaa ggttggagaa tagtgctcta 92700 gggcagtgaa catcaagttc tcatattttc caaaacatct ttaatttaca tgtgcaagga 92760 ggtgaaggaa tagtaaattc tcaaatacta atacattttc attcacttca atgttataag 92820 atacaactta ttcagggaag acggcagctc actgtgatta agcagtgtct ggatgctgca 92880 catccctgta tcactgggaa tgattatttc taaaggccca gaattcccag accactgttg 92940 ccataacttt ccaaagagct tcagttttgc tgctcttgtt ccaggtgtat aatacttaac 93000 acattgggct gtccaacttc tgtaatagca tttttgtttt gctttgtttt gttttcactt 93060 tctctgaaac tacctcatag ctctttcctc aatacagatg gatttggcag catttcagat 93120 ggaacaaaca tctgagcaga tacttcaaga tttgggcatt atgcaaagtc attttcagcc 93180 aagtaatttt cctcacaatc tggaaggctc atttgtgatt taattttctg acattatata 93240 atttaaaaaa ttaaaaaact tgacaggcag ccacaacctt atctaaatat cttctaccag 93300 aatatctgag attaagtcct ggatatgaca tttaaatttt gtcctgattt ctaccccaat 93360 gtttaaatcc aagatattaa cattttcata aaactaagtt tatgatgatg gaaaaaactg 93420 cactggtgat aaaagaagtg tggcttttag aatatcactt ttttttcctt ttgcttaggt 93480 ttttctgatt gaaatctaca cattgataaa acttttgcta ctattttggc agatactata 93540 tatgaaaata aatggacata aataaggtaa tgggatagct ttggatgaaa aatgtgtagg 93600 ttatgtgggc tagtcaaaac agatgcaggt gtggctgtaa taagcaaagc tgcttcaagg 93660 gaagtacaga tggcaatcaa atgtacagtg attactgcag cttaaaataa gaatactgaa 93720 tgagtgcaca tcatcaatat aataaaagaa acatttaaag aggagcaaca gttcatcaaa 93780 gatctactag ctgacgtgct aagaacagaa gaaaactaaa ctcaacaaag atgacaaagg 93840 attaattggc tccgttactg taagcttatg gctttcagta attgattctt ccccttttca 93900 ctaaaggata caaatatttc ccaggtcttg aaaagtatca taacagagac cataaactct 93960 aacactcaat tttggttgaa aatgacattt tggttttctc tgcttaataa gttaagcagt 94020 cactgctttt agcctgtgtt ttaacaagag aactgcactt tgctaccgtc tctgcagcac 94080 tgaatttctg ttttggagga gacggtaagt ccctcctcac aggaatcgca aggcgtctct 94140 gccctcccac ctctcatata tgctgtgttc tccatccctg cacccttaaa gacagtcttc 94200 atttaactgt ccaagtacaa ccacctaaag agtgaaattg cattgaagtg caaaatgagg 94260 tgaaataaat gatccatgtt ttactgttca gtgaaaggta gtgatctgac aagctcctca 94320 acaaatatgt atcaaagatt caattttact tattaaaatg atttacatgg gtcaaaccca 94380 tgcacctaaa accactgcag taattcgtta acccattctg ctttgggcga ctaacatttg 94440 catcaacaag ggcttgattg tggatttaaa gcattataca gcactagact tcactggagc 94500 tttgcatgta aagctccatc tgggcagttt tccagctggg taatctcaga ggactgatgt 94560 ctattcctca ggcactagtt agactagaca ctcttgttta ttcatctctt tcatttgcta 94620 ggcctaccac agaccaccct cttccatcct atgccttctt ttaacctaac ctgagacaga 94680 gctgggaagt ctttgccttg gcgtggctgg aaatatcagg gagaagaaat ttggaagaaa 94740 tgttagtcaa aatgctgaag caactcttct ttcctcccta ccaaaggatc tgaaagacta 94800 attagccttg agagtgacat aaaattccaa tgccagacat cccagcttta tgctcatact 94860 tatggtaaaa tatccaggat ataaaatgaa tcattttccc ttgatgtata aacagatgtt 94920 cattcagtca cctgtgcaag taaatatcca ccctatcctt aaccatgctg tttctaatag 94980 tctttggaaa acacacatct gatcatacca ttctcttcct ttaagacttg atgcttctct 95040 tatctagtag ctttaaaact gttggagagg gagtcagtag ccaatggatc taattttact 95100 catgttgctt cttttttttt tttcttttgt aaaatctatt ttgaacaagt gcctcaagtg 95160 aataataatg gctcttattg tagttattaa tattactatt atggtaatgt tgataactac 95220 ttaaggtcat gtaaagctgt gtcatgtgta gttaattact atatattaat attacaatag 95280 caatatgaat aactatcatg gcacaccttt acatgacaca catacccctt cgtgattttt 95340 gcccagatgg tcttttgtcc ttattctcct ccctcccaac tcatcccagg taactggctg 95400 tccagaatgt actatgcacc ctcaagaccc catctctttg tgtgctgatt tattttcctg 95460 gaccatgtcc ttccccttct ctgcttgatg aactctaaga ccaaaagact tctctaagaa 95520 ctgagtcccc tactttgtgc tcccacagaa tgttgtaatt atgtcttaca tttgtctttc 95580 actttccttt cttgatagat tgggccatat tttatcctta aatttcaaaa atatagcaat 95640 attcctggca catagtaggc attcaataaa ttcttcatga atgaataaat gaaaatgagt 95700 aaatgaatct attcttattt aggttttctt aaatatacgc tttgcatttt cctacaaagt 95760 ctagatcgat atagaaaaac aaagcaaaat ggaaagaaac attttaattc tgtatttccc 95820 tttgcttctt gatgcaaagc atctttagaa aagttcagta taactctgtg cttgcctgag 95880 aaatatgtat aaggagataa tttatatagt gtactcgaca caaaaaacaa caggacaaga 95940 agccatgaat ggaaactata taaccagaac agaaagaata gctcctgcat tttcaggaga 96000 aacagtctgt tagggaagca gtcttgagac ccagtcttat tttttagaat agttaaatgg 96060 caacataact cagtacccgt aatccccaac ctgattacag gctgtttgtc cactattttt 96120 ccatcttcaa tacctcaaag ctctgacttg ggccccttat ttattttaag aatctatgct 96180 ccagggtctc tactctggct accatcagtg gtctggatct gaaaagtttc tcgctaatcg 96240 ctatgcttca aagcagataa gatagagaaa aaaagatatt ccttaaattt tttagcatct 96300 aaattgtgaa tttcgtatgg actctgtcat ggaaatgtat gttcatcctc tttgaagatc 96360 cacaaattat tctgtattta atgggccttt gtaggctggt ctggggaacc tatcacagat 96420 gatttaactt cactgtgaaa atatgttttt tggtaatttt ttatttagat ataatgccac 96480 gtttatagaa aagttgcagg aatcgtacaa aaaactccca tacaactttt caccaagatt 96540 atatacattc ccctcatttg ttttgtgtat atgctaatac atcacaaaca cacaaaatac 96600 tttttgaatt ctgattgaat tataaacttt ttgagtacag attgtaagca aattgaggtc 96660 tgctgaaatg tttgatcaag actacattcc atttcatgct tttacatttt ctttatttct 96720 attatttccc cataataaga gttcggttcc agaaagaaaa atgtatttac attttttttc 96780 cttgtaagta gtgacttaac ttcatatatt tgtgaggatg taactatact ttctcaaggc 96840 ctatggcact ttccaataat aggctgagtg gtttattgag tcaaagtcag tcctgtaaga 96900 tacggggtta tcacttagta aacagcatca ttgtaatatc tatagtagta cccttggaat 96960 actagaggcc aatcagtaat gaatatccct gatgtcacca tgggctagcc tgggtactaa 97020 gggaggcagt taaagaagtt ttaaactgag ttatttctca gaaccccaaa taagagctca 97080 gcctatctat ggctctgcta atattgattt taaaactata acgttcatct ttttcttgct 97140 ttaaattcaa gctgttttta aaatcaaatc tttatcccca accaaaatgt agatttttta 97200 gttcaccaga gaacttcaac tgacctaact aaaactaaaa gttcctagct gcttaaatgt 97260 ttatgtaaaa caaaaagaaa aagttgaagt tccggaaaaa atacttagac atttccataa 97320 tgtagtcact gcctcatttg cttcatcaat actaacagtc gtttttttta agtcaagata 97380 tttcaagtag aataaaaaca cttcatatat gtatatttaa tgagacaaaa acttgattct 97440 tagttcatag acatacacca aaatgttcag cacaaatact caagaaaaat gagatatcca 97500 ccaaaagaac aagtcttatt ttctacttcc atacttagat tttcttctta aaactttaaa 97560 cagctcagca tggcaataga agatgtctgc tgaagtgggt ccatattgtt ccaagtgtgc 97620 ctgcacgtgt gtttacgtgt gtaaatgttt taccaatgtc ctttatggtg ggtcccctgg 97680 atgattactt tactcccatg ttgcttccag ctaaattaga tttgaatagt attactgcat 97740 tttaatagaa gatataaacc tcttttattc tgaatacttt gctattatga tagtaaaatg 97800 aattaatagg aattaaattg ctaacatgga ttaaaatagc ttttaccatt gtagaatttt 97860 aaaaaagtca aacaaaagaa atatttatta ttataacaca cgttactatg gagagtattt 97920 ttaacttcca gaactaaaaa taatatttgg tatttacaga actattttat attcatattg 97980 catctaaccc ctaattgctt attttctaaa aagaaaaaaa gaagaaagtg caatagagaa 98040 aaaaagtcag cctatattca aacatatact aacagttaaa tgagaaaatg caataaaatg 98100 cagagttgaa agtttgaaag aactgagggt gtcaaagaat gtaatttcta ttcaaaaacc 98160 ttttttttga cgacactatc acaaacacac tttaataacc atggcattcc accaggatat 98220 gagattgcag gtgactccat cagtttgtac aattgactac aattatctga tatatgtaca 98280 aggtgctgaa ttttgagtac aggtagtaag tgctggttat ataatagaaa tactcccaca 98340 aaagtagctt tttaaggttt tttttttttt tcttgaagag ctgaagtact gaagtgagta 98400 catacatttt gtcatgttaa acaggagaaa gattgtgact tggctcctga aattttaata 98460 ctgcttgtta gccatctaca atccctaaag tgctttcaga ggctctacac atcttcaatg 98520 agtcaaaccc caaagactta taaataggta tataggttgc taatctattc ctgatgcttt 98580 gcctaaaaaa ttggcattgc catcttccac atctttcttt tggaagcatg gttttgagtt 98640 tgttccttgt tggtaagcat ccagaaaatg gcagattccg gggatttcac tagggaagtt 98700 tggtggaagt tcctcccttg atcgaggggt aaacacgaaa ccaggacagt ctttgagtaa 98760 tctgaaaata ttaaaatata atactgacat aaatgtttgt ccactcaaaa aaagaacaca 98820 acataactaa ctgaatgtta ctgagaatat ttataaacca gtgattttag gtgtgagtga 98880 cagaaaaagt ctaacgagat taacagacat cttcccagaa gtacaatgaa gaatcaattc 98940 acaacaagaa aattgtgtca attaatgtat ttcacttaca ctggaaaaaa ttataaaata 99000 tttttgcaaa aataatatcc acacaacagt gcaatttgca gtagtgaaaa aataatccaa 99060 agatttaata gtagcaaagt attcacacat aaccataaca gtattaagat ttcctgagtt 99120 gaaagaggta agatgtccat ttatcaattc attaattttg taaatattat gctaattgtg 99180 ggcataaaac gtaatcatgg gcaagaaata gtgaaccaaa attacaatga caaaaatgat 99240 gtgagaacaa cttttgaatc tttcaataga atttttatga attctagatt agtaggactc 99300 aaatgagatt taattgtaca tatcaccact ctacttacta attgttatta gcaatatttt 99360 ttctcctgaa attcatctgt ttaccacatt atattgtttg ctattttact acttgttcat 99420 tatgctaaac ctatttcaaa ttctaatctt actgattaag ctattggcat ctctttccat 99480 ttgcttccct agctttccat caaatgagaa cttaataagc cttctctcta acctaatatt 99540 caggccatag atgaaaaatg ttgcatggct gtgggttctc aaccaatttc tacagaaaag 99600 catttattat atttacccaa acagaccatg gtcttctact cactagtctg taatgtcatg 99660 tctacattaa atagaatgat gatgttgaat tagattttgg tactgtgaca acatctatgt 99720 attgaagaaa ccaactgaat agaaatctgt ctcaagaatg acagtttaat tttcatcagt 99780 acatcaataa atatgtaatc tgctaaacaa taaaactaat gaaaatgatg ccttagtcat 99840 gaatttataa atatttttga gtgactatta tttgagaata cataatgaaa aggtatagaa 99900 ttagacagta ttaaaagtat aagcttcaaa gtctgatagc tctagcagca cagatgtcca 99960 ttatccagcc tttaaaatgg ggataataat agtacatatt ccttaggaca gttgtgaaaa 100020 ttgaataaat caatttttaa aaactgacta agcatcaatt agaaatcaca ccacacctat 100080 catattggta acagtaagtc tgacaatatc accttggaga atagcacact tcatacacta 100140 ctggtggagc atagattggt aaaatcattt tgaacagcta attaatgctt ggtaaagttg 100200 aaactgggta tagcctatgg tctgggaata ccatttttag ttatattatc taaagacata 100260 tgtataaaga tacctattga atcacttttt gtaataatga aaaattggac acactctaat 100320 tacccacagg tagaagatag agtctggctc attcatactc tagaatacac attatatcac 100380 atgcacatat agtaacatgc tagatctggt tcatactggc ttgtgagagc caattgttaa 100440 aattcaggaa ctttgtaaga cagttaagcc atccatagcc tgaaacctgc cagagtggac 100500 atagtaacac cacagaaatt ggcaaatgct ggaatcatgg attctctacc tcatcctcca 100560 aagctggttt accagcaaac cactccatta gcaatagtta aaatgaataa tctagattta 100620 tgactagatt tacattttat caaggaaact atctcagaaa cataatactg agtggattca 100680 aggcaagtct taaaaggata tggatatgtt cactgggaat ctattttcct ctggctttat 100740 ttttctatct ggcaggacct aaatgaaagt gatattcctg cctctctcta cccattttcc 100800 ttcctggaaa gcagaagtga catcttattg atgagttgtg gagaagctgc tgaacatgca 100860 tctgtatttg ggagactgtc tgcagagagt acatattctc agctcctaca agctataaag 100920 cacttaggat ccattgtgtc agagcgtgta acagggacca tgtaagagtt tctatttata 100980 ttttattata ttaatatcta tttactttac attttatttt tttataaaac agaaacctga 101040 aagaaatatg gcaaaatgtt gatacttaaa tattagtatt ggctacatga gtgtcatctt 101100 gtttgctttg tttttttata tgtttaaaaa ttaaatacaa aaatgtatgc aactataaga 101160 gtctaataaa taaaaatgta taaaactata ggtgattaaa aaaatttatc tattattttt 101220 tctatctaca gcaactcaac atttcctact aaatacggta atgacaatta taatctccta 101280 tttaaatgta ttatatacat atattcagcc tttagaagtg gctttaaagt gcatcatttg 101340 aatttattaa gggcattgct ctagattgca cgtctggtaa gtgctgtttg tgggactcta 101400 aggcatgtct tctgaataca acttctatgc tttttaaaga caccttaaca gacaaacata 101460 attagaatgc tatggagttt gaaatgttca ttatattgtc tttttaaaaa atcagaatat 101520 ataagaaaaa ccatttagaa aaacaaacca tttcgatgaa agcttgatga tagcaacacc 101580 caaaaaaggc agcaacagcc cagatgtcct cataggtgga catttttctc tacaccttct 101640 tcaccagaca ccatttttcc gcaaattact gcatcaatgc tccaattctc tgttatcttt 101700 cctatattcc tttattcact ttgttccttc ttcttacaga agtcaattct tgcttctcta 101760 gcacaatgtg ctcagacttg tcctacaaat aagagattct aggcctgttt accccacaaa 101820 ctggacagca tccatgtatc ctccccactt gccatttttg tttctaatga tccctggatc 101880 accacataat cctctaacat tgacccactg acctggaact aatgtagtag acctggatcc 101940 actgactcac tgatctaatg tagtctctac tagtctctat tagttttcct tacttcaaga 102000 atgcaaacaa aatgtcgaat attatcccct aaaattaaag taaaactcct gacaatgttt 102060 aaaaaacagg agtgtataac tgtcattaca ctgctacatt ctgaatcact gattagaatt 102120 tggaagtgga aatttgaaga tacaggtttt tttttggtat aggtatgtat tctgtgtcct 102180 aaatttacct gtatttcaga aatatatcat ggaagctaca ttcaatatta aacagattat 102240 tttattatta aagaaatatc attccagaat ttaatgtatc atcaataaag ctctccaaag 102300 aaaactgcat tttgtgcttg tacaccttct ttagcagccg cttcttgtac tatagtttga 102360 gtaaaactaa aattatgccc ctaccgttga ctaaatttcc caaagcattg gatgtttaat 102420 tgaaatttat ttatattatt ttaagttttc ccagcatatg gaagaaaagt gaagtatctc 102480 tgaaagaaag gaatttaaaa tgttgttaaa agtatttaac attaagtatt tatattttgc 102540 ctaaataaga cagtattaac tactgctttc ccttaggcaa ggcataaata tctcatatac 102600 gagaataaaa agaaagtaaa ctattattga aatcaataca aaaggaaaaa agctatatca 102660 tatttgtcta taaaaagtag aaatgacttc agcaaatgag ctgaagttca atataaatta 102720 aattaaaatt tcaatgtcat tcatgcatgt gtatataaac gtaactgaaa caaagacaga 102780 aatgttaata gttactatct ctgggtaatg agattatgac ttattcctat tacttacttt 102840 ataatttact aattttgtaa attttctgaa atgggcatgt gttaccacag taataaaaaa 102900 tttatttaaa agataacacc atctatgcta taaaaaccta tatgtgcatt tctagtaaca 102960 ttgccaatac agtgaatcta agacttaagt catgagcatt gtcctgttgt cagaaatata 103020 tgtcaacatg ttgtggtcct ctgctgaaat acatgagatc ctgaattata ttttgaagaa 103080 aaataagtag ctaatatcag tgccccctaa catgactcaa gaccaccaga ctttgagata 103140 caaaatgcaa gaatttaaaa agtttagttt catactgtta aaaacaatca atatagaaga 103200 aaaaggaaaa tatgcttttt atatgtagca gttttgtttt aaggtaggat tcctcagtat 103260 gcacatatac acataacaca tcacttaaca ttgaaagaga aacacaggtg tcagtgaaag 103320 gatgggcaac tgactcctac ttaataggat gattcctctt aatgcctaca gagagagtag 103380 aatcatatta tctcaaggca tggattctta tccctaccta ttatacccag taggaccata 103440 ggccaattac cattacaatg aaccccaggg atttttccaa ttctcaggtt atactgagtt 103500 gttttcatac ccaggagtca acagttaacc agtaaatact tcttgctttc cttctgtgtt 103560 tcacgcgctg atctaggagc tgggaatatg gaatgtgtac cacaggaagc taacattcta 103620 aaaagggagg caggcaatca acacataaat aaatactgca taagacaatt tcagatacag 103680 gtgtgaactg agaagatgaa ataggataat gagagggtat gtgaggagaa ggttgttgct 103740 atagttatga tagttaagga gtgtctcttt aaagagagga gaagaaatga tcactgtaag 103800 gtcgatggtc tgagtaacta ggtggatggt gatatcattt taaggtgatt ggtaagcttg 103860 cctgggtggg aagtggattt catcagtctg tgtgggaaat cactcattct aattcggtcc 103920 agttcatttt aatatgtgaa ttagtcatcc aagtggaaat atggaatagg atatatgagt 103980 ctgtagctct gcagagaatc acaggatggg cgaaagattt tttttaaaaa atagttagag 104040 tcataaaagt tcagatacag aagataacag gaaaagaggc tctgagcacg gcaacaaagg 104100 tttggtaggg aaggagtacc aagagaagac actgagaagg tgtagacatg gggtagtgag 104160 aaaactaggt gaatgtggtg ccaaaaattt aaagaagaaa atgtttaaat gagggagttc 104220 tcagcctatt caaatgcttc tgagaggata aggagagaaa actgacagtg attttatttt 104280 agcagggcac aatgatgacc ttgataagaa ccgtttgagt ggagtaaaca ctgactggag 104340 tgagttgaag agaaaataag aggtggaaaa aatacagata gtgagtatag acaacttctt 104400 tcagttttgc tataaaacag agcagaaatt gaaaattgta gcagaagccg gacttggaaa 104460 caaggaattt tttatttttc aattcagagc tttaatagat atggttgtat tctgttggga 104520 ataatcattt catcaaagag aaattgatca tagagagtta atttccacag aaaagttatt 104580 gagaaggtga gaggaaatag cactgagagt aaaactgtat caaccagggg caggagggga 104640 gctgggccaa tgtaatggga acaaagaagg aggagatgag tactggggta gctctgctga 104700 gagaacctac agcacaaaga tggcaaagct cctgtctgac ttctattctg gtggtgaaat 104760 atgaggcaag attatcaact gagagtctgg gctggaaagg aaaggatgct ggtgccttaa 104820 gaagggagaa gaccaggtgc attggctcac gcctgtaatc ctacactttg ggaggccaag 104880 gtgggcagat cacttgaggt caggagttcg agaccagcct ggccaacacg gcaaaaccca 104940 gtatctacta aaaatacaaa aattagccag acgtggaggc gggcgcctgt aatcccacct 105000 acttgggagg ctgaggcagg agaatcactt gaaactggga ggcagagctt gcagtgaggc 105060 aagactgagc tgctgtactc caacctgggc catagagcaa gacttagtct caaaaaatat 105120 aaataaataa ataaataaat aaataaataa ataaataaat aaatgcagaa gaagaaggga 105180 gaagatgcta ttaaagagtt atctctgaga ataggaaagc taacttgctt agtataattt 105240 agctactgta tagatctgaa aaactctttt gttacttaag agtttccaat ataatgagac 105300 aaaatgtaca tgaaagatat atttctagtt accacggatt ggtttatttt ccggttaaag 105360 gtggataagt gataacaaaa tggtagattt tattgactca ggctgtatac tacatgttgt 105420 caaggcgaca gtgaaaataa aggttcttct gatgctagat gacacgtggc tcctctgacc 105480 gtgttaacac ttgctgccag tccataaaac tgcaatcttt gaaaagtgct cgatgaccag 105540 ctagttttcc tctggtaaat tcttccctgt atgataggga atttgactgg ctgttgtcct 105600 tgattgcagg gagggagctt ctaaaactct tggatttttt gagttatagg agtatctgtt 105660 ttccatgagg cccttggatc acttttgagt ttatattaat gagatgactc agaatggggg 105720 cagggcccca gaaaaatcaa tcttgtggat agaaggttgg ggttttgtgt tagcctaacc 105780 tctggggagg agagtggggg tggtgactga gttcaatcac atggccaatg attcaacgaa 105840 tcatgcctac ataatgaaaa caataagact ccatacacca aactcaaagc tcaatcaggt 105900 ggagcttcct ggcttgtgaa cccactgagg tggctggagg gagataagtt ctgattctat 105960 ggaagctctg catttgggac cctcccaaac cttacccaaa ctgtttcttc atttggttgg 106020 ttctgatttg tatccttcga aataaaactg taatagtaag tatagcaatt ccctaagtat 106080 tgtgagtcat tttagataat tatctaactt gagaggttca tgggaacccc caaaattgta 106140 gctcagtcgt ctaaatgcat acttaggtcc tgaagggtgg ctggcatctg aattaagggc 106200 aggcttgttg ggttctgtgc cctgtaactt gtggttatga ctgtgctgag tctgggtgtt 106260 tactgccaga actgcactgc agtgtactag ttgatgtcag aacacctatc aacatactaa 106320 gagtattaat atgtgcttct aaaagaagaa tgatggaaga ctatgtaata tctgtcatgg 106380 atttatggct gggccacaaa cacctcaggc aagtcattct taggaggcta ataagcagtg 106440 taaaaaagtt aaagtgagga tgataaaagt tatcacgttt cttttttcct tacgactaag 106500 gaagaaaaaa gttatgaaga atgaatcaaa tcagtggggc aaatggcaat tgtatataaa 106560 taaaaacaag agattttagg tgtttaacat aattactacc tgtaagttgt tgggctgaca 106620 ttgatttttc gtggtacact gcaggactca aatttgttag ccagagtgac attgtttcca 106680 aaaagacagt aacggggcat tttaactcca acgacgccag caaaaactga tccagagtgc 106740 agtccaattc gcatctgaaa gcaaaaaata acatgatgtt cccagtcact ggtaaaacac 106800 ttcctactcg tcagtgtcgt ttgaataccg tctaaattat tttaccattc accctcagga 106860 caccacccct ttcctcaaaa ccttcagcca acttccctct actcctcaca tgcaattaaa 106920 aattactagt gcagaatcaa cagtcacact tcttaactct tttccattta ggtttattac 106980 acaaaccatc ttaattgtgt ctgtcttcaa catctgaaac aagtttccat gcacagggat 107040 catagtaacc acagcaacat agcaaggggg atatgagctg tttatgtgaa actgccacct 107100 ctcattcaag tgagatagac ataatttctg gactcctgga atcattttcc caaacactga 107160 caaagaaggg caaatgaagc tagctctaag gatattaaat atatctttta gacattttac 107220 ctcaaaatag ctttatacca aacactatcc cactgttcta atgacttaca actgaggaaa 107280 tgaaattgta atatttaagc agaaaatgac agaggaagca gaaagtggaa ggtacttgag 107340 tccaatgacg atgacaatgt ctatatacat gaaattaaaa atccatactg atgaaagatc 107400 agatttgtgc catgcctaga tataagaggc agtctgggat gtgttgtggc aaaattttag 107460 gaattctgga agtctccaat gatttggaaa gccactgaga cagaatgttt tattttattt 107520 attttttaga gacagagtct tgctctgtca ctcaggctgt agtgcagcgg taccatcata 107580 gttcactgta acctcgaacc cctgggccca aatgatcctc ccatctcagc ctcctgagta 107640 gctagaacta caggcatgca ccatcacacc tggataagtt ttaaattttt tgtagagaca 107700 aggtcttgct atgttgccca gctgattctc aaactcttgg gctcaagtga tcctcctgcc 107760 tcaacttccc aaagtgctag gattacaaat gtaagtcacc ttgcctagcc aaagcaggat 107820 atttaacaat ggaatcttca aaggtctgat tacttgatga tcttaaaaga ttcacaaaca 107880 cttaaattac tgctactaca aaacaagcta aagtggagat attttaaatg atgatgatac 107940 catgttaatt attttcaaac tctttgttta ctcaaccatt ttaaaaagtt gtattagatg 108000 tatgcgtttt gaatgaagac caagaactca gtaacttgga tataatttag aaatcagaag 108060 aattcagtta tgtctagttt taataactaa tttcagatat ctgctagaaa ctttccagac 108120 tcatttactt ttaagatgtc atatgagttc tacaaaattt tcaaacaaaa gcactagact 108180 gtgaatcagg aactttgtat tctaacaagc tctactagta actggtagtc tggcttcctt 108240 aagtctcttt acttccctcc gatcatgttt tcacctataa aatgcagggg tttgattatt 108300 tttaaagacc tttcttcatc aaagttttac tttaacaagc aaaacttaga tgtctgttat 108360 atatctggct ttgtctcttt taggagaata ttaatttaca catttagcaa gtgatagata 108420 gatagataga tagatagata gatagataga tagaataaag ctcttctgct cataaatatc 108480 aactatgact tcagcatatt tagattgagg aatttttgct ttctgtgagc actcttctgg 108540 ttaaaccagt ttttcttcct atacttagtt actccagctt attatatatt aaatttacca 108600 tagcattttt atcttgtttc ataattaaaa tgtaaatata ttctgctcgt aaaaaccgaa 108660 acgatgcaga caaggcaagt gtccctccta gtccttccat gccgtgctgc cttcctcccc 108720 ttctccagtt ttgaatctct caaccctaac attttcttac agagcttttc aaattcatca 108780 gcaagattta aaacacaacg tgtttaagac acacatcgtg ccctgagcat atctcttttc 108840 actggtaaca gacttccctg tgaaataatg aaaggcaaag gcagaattct ggctcccggg 108900 aaaagtttga atgggaacct actaagggca tcttctattt gagaccactt gtggcatagc 108960 ccatttctca tcataagctt ctaggcttag gaaaggaggg gtactagaag tttgcttccg 109020 tgttcacttg tcacctactg actttcacgg ttacactcag cccactggtc tctgtcacca 109080 acccacctgc tgctttctaa gcctgactgg atggatgtta gtgaggaaat caatgggaat 109140 aaatgaatag atgaatgatt tgcacatgtg acgtgctaaa ggaaagcata gagtttgaaa 109200 aataaaaaaa gaaaatattt tctattcttt cacttcaatt ctataccaag aggtagcagg 109260 gcaagaagca gaaaaagcct gggctttgaa gtcacatgga catgactcca aatactgaca 109320 cataggagac ttgactgctc gtgtctgcca gtgtagatgc ctctttaaag tgttattata 109380 aggattaaat gagacgatgt taagtcacaa tatgctcctt tctgatacat gttaaagttt 109440 ccctactaaa aatactctcc actctgcctc tgtgatgttt caccggtatc ttttagggac 109500 gtcagatatt tcccagggct gcaggttagt atgcataaaa aggtatgatt aatgattccc 109560 tttataggtc ttttaaacta atctatcatt acccaggtaa ctatctttga aactagctat 109620 aaataattct tccaacttag aaaaatctaa aaatacattt ctgcaaataa caaaaatgcc 109680 ataaattccc ccaccccaaa catacacatc ttaattataa aatgtatttg tgtgaaaatc 109740 acttaaaatg ttgttaatag tctagatatt tttaacattt acattgatgc tacaagcccc 109800 ctcaaacaaa ttagaaagag aaagaaatat atagtcctcc tctaaaaaaa tgcaattatc 109860 gaaaataaat ctgaaaaagt cagatattgt gatgctttta aaacatactt gcaaattatt 109920 ttacactcct cctattgatt cttgggctct gagccctctc tcttctgggc agacttgtga 109980 ttgctctggc tgatagagtg cagtggaagt gttgcggtgt gtcttccaag actaagtcat 110040 gcagcttcca ccagtcttgt tcagtgggac atgaaccacc tcataaaaaa tacatattag 110100 ggcatcacat gtaacgctaa caatcccagc tgaacccaac ctttcagcca tccccaccaa 110160 ggaatctgac atgtaagtga agcagcagtc ttggaatgaa tcctccagct tcaatcgttg 110220 caatcccagc tattccaggc accctcaccc atttcggtat tcccagctga ggtcccatag 110280 attgtagagc acagacgagg tattcctgct atgacttatc caaatttttg atgcacagaa 110340 tccacgcata ataaaagttt gtgattttat accattaaat ttagaaaagt atgttgcaca 110400 gaaacatata aatgggatga caatagactt agaaaatagt tacgaaggct taaacatatg 110460 gcttctatta aggttaaaga gagttaaggg taaatctcca aatatagttt tgtttttaaa 110520 tttcttcttt cttctctata tgttttatag cataccggat aagattttac aatacctcaa 110580 aggcttattt ctgaaggtag tcaactgagg actgtcctat aaaaattaag gggcagattc 110640 actgtgagac aaaggtaatc tgcttatatt ctatctgtta aaccaatgcc taccatgtta 110700 actttatttc ctcatgtgtg gtgagatatt agtctggaat ctggctcatc tagaaatttt 110760 gaaatgtcac gtgccttgga aaatattggt gaattaacag tcaacatcca ttcaaatgta 110820 atatcctctc ctccaggggc tggaaaacta aaactacatt ttctaaacta acttgcatct 110880 tctaaatgca gttaggcttg gtggggggga tgcacagttg catgagattc ggcaggcaca 110940 atctccacta taactcaaga acaattgtgg aagcctttgt gttttctata gcatgcttag 111000 tagagagtcc attttcccat cactcatttt gtaagtcatg atgcaggatt gccatcttgc 111060 tgattattat agtagatgtg gttttgaaga ggnnnnnnnn nnnnnnnnnn nnnnnnnnnn 111120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 111180 nnnnnnnnnn nntaaataaa ataggatgga aatatgccaa atataatgtt ggttaaatct 111240 gagttgtgtt aaccatgtcc aaaaatttca taggtttttt tgtgataatt ctttaccact 111300 taaactactt atttccttcc ttatccatta tttgcctgcc tttgagtagg ttttgctagg 111360 gatatttaac tgcaggtaca acttttccaa acttaatttt gttttttcct tggcattcaa 111420 tctccatgga tatgacatca acaagatttt caggtacttc cttccacaaa cacatcttca 111480 ttctaggatt attgtctact cctttcattt atatttaagt ctataagtaa agtcataaat 111540 cttttattat aacatataac ttcagtggag agattaaatt tttattttat ataccatacc 111600 tcagttgatt tgttaacatg aacatgttat ttctcaaacc ttccggcaca gattttctat 111660 ataacatgca tttcctgcta ctatggtagt taccagaggc ttatgaaact tcaggttcca 111720 tctgcaatag ggggagcata accataactc agccttcaag aaataaattt tttcaacaac 111780 ataagatttc aggtgtgtta gtccgttttc acgctgctga taaagacata gccaagactg 111840 tgaagaaaaa gagatttaac tggacttaca gtttcacatg gctggggagg gctcagaatc 111900 atggtgggag gcaaaagaca cttcatacat actgtcagca agaaaaaaaa tgagggagat 111960 gcaaaagcaa aaagccctga taaaaccatc agatctcatg agacttattc actatcatga 112020 gaacagtatg ggggaaacgg cccccatgat tcaaattatc tcccacaaca catgggaatt 112080 atgggagtac aattcaagat gagatatggg tgggacacag agacaaacca tattactagg 112140 tatcattacg aagcactgac catctacttc ccaaccaatt gaatgtacaa tgggaaaatc 112200 ccttgttcat tcgcagcact ctggccagga aatagcaatg ttacatgagt gttcttacca 112260 gagaatcatt gacattggct tagtccgtac ataatagtcc tacaaatgta ctcctctagc 112320 ctgaatctct atcatcttga gccactgcct cccaactttt aaatacactt tttttttttt 112380 tttgagacag agtctcgctc tgttgcccag gctggaaggc aggggtgcga tctcagctca 112440 ctgcaacctc cacctcccag gtttaaacaa ttctcctgcc tcagcctccc gagtagctga 112500 aattgcagct actggctaat ttgcacccct ggctaatttt ttgtattttt agtagagatg 112560 gggtttcacc atgttagtga ggctagtctc gatctcctga cctcaggtga tccacctgcc 112620 tcggcctccc aaagtactgg gattacaggc gtgagccacc ctgcccagac taaatacact 112680 tttaaaagca gttttaggtt tacacacaca cacacacaca cacacacaca cacacaaaac 112740 tgagtgaaaa atacagagct cccatatacc cccttaacct accaccccca gtttccccta 112800 ttattaacat cttgtatgat tgtggtactt ttgttgcaat cgatgagcca atattgatgc 112860 cttattacta actaaagtcc atagtttatg ttaggattca ctctttgtgt tgtacattct 112920 atgggttttg acaaatgcat aattacatgt gtctaacatt acagtatcat acagactagt 112980 ttcactgccc taaaaatccc gtgtgctcca tctattcatt tttccctcct tccctcaagc 113040 tcctgtcaat cactgatatt tcaaaattct ttatggtttc aatagttttg tcttccctgg 113100 catgctatat cggtggaatc ctatggtatg tagccttttc acattggtat ctttcactta 113160 gcaatatgcc tttaatgttc tttcatgtct tttcatggct tgatagctca tttattttta 113220 tgttgctgct ctattattgc aatcaagatc ttatcaaatc cttcctgact gaggatatgt 113280 gctctttcca aggatgtttg ggaaggggat gctccattag tgtagaaaca tctattgtcc 113340 cttgtatctc tacattatct tttcaggcag cttccagtaa tgccaggtcc cctgggtcct 113400 ttatgaatat tctgtgggtg aatcgccagt gataaatgga tatacagaag tatgaatatg 113460 atcaacataa cttgagggaa aaaaaaccca aattgtgatt ccaacctgct cttcttttag 113520 gcaggtcatc aagggatact gattgataca ctttctttga ataactcaaa cagagttgtt 113580 gggttaactt cagagtttgc cttcaggtca cttctccact tttggggaaa agcaaatcca 113640 ggggttatct cagcatggat cctgacccat accccacctt gagagaatta agagtcttaa 113700 ctatcttgct ctgaggctct ctctcaggaa aagaataata gcatcctttc ccatttgtca 113760 ttgaggtgct ctcttcccat ctctcacagt ttctcccttt ccattttcac ctgtctcatc 113820 tcagacttgt cctcttcagg tgagattttt ctcattcagt cattctacca tctacaggca 113880 agtagcagaa acagccttcc ttgaacatcc ccttttgtcc attttcaaga aggagtactg 113940 ttagaaaggt aaagattact ggactattcc taccagaggc actgggccag catttccagc 114000 cagtcctcca ttagccacat gatgtggagt cctcactcga tctatctgtt cccttggtaa 114060 tatttatact ttctgaagaa tcagcataat gtttgcatgg tgattcttcc caggatgcag 114120 ggcacacaag tgcaatccca gagctggtac aactctatga agtaaaataa gaaaaatgtt 114180 tttgaggcga gaaagtcaga gagtatcctt ggaccttcat cctcatcccc atatcccaga 114240 aactgtttca aaggtgttcc tgctccacta tccctcatat tcagataagg tatttactca 114300 ggagtcagct cagctcttca tgcctctagc acattcttcc catattttag taaaggtgtt 114360 ttcattccac tttgaaggtg gtaggggata taataacaaa caatgttaag cttcttacct 114420 catgtaggta catactaata gcagtggcat gtcaccttgt aaataaaatg tagttggtgg 114480 gaaccaaact gatcgacaat ctgtttttcc aaacctttaa ggaggcttaa atttaataat 114540 ctccattctc agctagttag ctcttccatg acatcaaccc cattttatct ctttcaaaat 114600 acagttgccc tttgaacaac atgggtttga accgcactac atggcttttc ttccacctct 114660 gccacccctg agatggcaag acgaaccctt gccctgtctc ctcctcctca gcctactcaa 114720 catgaagaag acaggatgga gggcttcata atgatccact tccacttaat gagtagtaaa 114780 cgtgttttct tttcttcatg attgtcttaa taacattttc ttttctctag ctaactttat 114840 tttaagaata cagtgtatag gctgggcacg gtggctcatg cctgtaatcc cagcactttg 114900 ggaggctgag gcgggaggat cacgaggtca ggagatcaag accatcctgg cgaacacgat 114960 gaaacctcat ctctactaaa ttagccgtgt gtggtggtgg gcgcctgtaa cccagctact 115020 cgggaggctg aggcaggaga atggtgtgaa ctcaggaggc agagtttgca gtgagccgag 115080 atcccaccac tgcactccag cctgggcaaa agagtgagac tttgtctcca aaaaaaaaaa 115140 aaaaaaaaaa aaaaaaaaaa gaatacagtg tgtaatacat ataacatata aaatatgtgt 115200 taattgactg ttggtgttat tggtaaggct tccggtcaac agtaggctac tagtagttaa 115260 cttttggatg agtccgtaat gatatgtgga ctttgcactg aacaggggct tggtgctccc 115320 aacccttgta ttgttcaagg gtcaactgca tatgtatttg tcgttttctt atcccaggaa 115380 tattggaaga taaattcatg agccatatgt gatacttaat atttcttaaa atacagactt 115440 cctcaagttc aaaaaatagg agcgttttct caattatatt ctgctacttg acccatttta 115500 tgttagacac aacaaaattt gtggagtctt tctatgaatc attcaaaata caaattgttt 115560 caagattttt caatatattg aaaaaaaggg gtaagaatgg tatcttctag ctgagtttat 115620 taaggggtgt ccaacctttt ggcttccctg ggcatcactg aaagaagaat tttcttgggc 115680 cacacataaa atacactaac actaatgata gctaatgaga tggaaaaaaa aaacatccat 115740 acatcatttt cataatctcc gccactacag ataagcaaaa aagttatttc attcaaaggg 115800 ttacacatgg ctataattag aatcaatatt tggataagta ggccgggcct ggaggctcac 115860 gcctataatc ccagcatttg gggaggctga ggtgggtgaa tcacttgagg tcaggagttc 115920 gagaccagcc tagccaatgt ggtgaaaccc catctctact acaaatacaa aaattagcca 115980 ggtgtggtgg caggtatctg taatcccagc tacttgggag gctgaggtag gagaatcgtt 116040 tgaaccctgg aggcagaggt tgcagcgtag gttgcagtgg ctgagatcac accactacac 116100 tctagcctgg gcgaaagagc aagactccat ctcaaaaaag aaaagataaa tagaggaatt 116160 gcaataaagt cagaagctgc atgccacatc tatgggagaa tttctccaga gtatttaccc 116220 aagattgaga tttctgagtc atgagatatt tgtattccta atgtcagtac catgaaattg 116280 cactggcctc attcccacag aactagcact tgcttatcat taagctggaa aacaatatca 116340 agacttttag gagttattct cttacatctg ggagaaattg caataccacc aagaataaat 116400 taggggatag attctttgac ttgattatta tttcttaaat atgtttgaga aatcttctaa 116460 aagtcacctt taattgaata ttctgagaca ccgaaagtac atctattacc cgagtgggag 116520 agagaacact gaaactcctg agtttcctct ctggactaaa tatgtaaata gaagtacttg 116580 ggaaaaaaat gagaaaatgc catgtatttt caagatagta cagatttaac tttattttca 116640 tttagattga tgttaagtac cacctgtatg ggaaacagat ttctgtccct ccgaaagtca 116700 tttacaatag aacactccag ataaaaggga aaaaaaagta tgttgaatct taatgaaaat 116760 aaaaattctt taaaacttta taaagcataa attatacata tcatttcttg aataaagaaa 116820 tattttgttt cctcatgtat agtttattta ttgggtctag tttacatatt tagtcataaa 116880 aaatgtctct tttccactac accaatattc attcattgag aaacatttat gagatgtcta 116940 ttacataatc ttgactgttc taggtggagg gaacacaaag gtgaaaaaga taattcctat 117000 ttctttattt catatatata tatatatata tacacatata tacacatata tatacataca 117060 catatatata tacacatata tatacacaca gacaaagaca aatagaaaga aatatatatg 117120 aagagataca tatctttaat ggcttttatc gaactctcaa aactatagaa aagtgacgta 117180 acaatgaatt agtcaagata aatctgaagg tatcatgaaa aggaaactat tttattctaa 117240 gtgaccatta acactcctct atcctatttt tatttcccag atgattctga ggtggaggag 117300 tccactgggt catttcagat atcttcagca aaaagaaaat gcatatccag tagaaattgg 117360 tgaatagata ttcaataatt tctatgatcg tgatcagact ggccccacaa aatagaccca 117420 gctgaccacc aagatctgct gtaataaaac caataaagat tgaatgtcat tatttttcat 117480 aaaaaacaaa tattattaga agttaaatta gttgaagcaa aatagagtca taactgagat 117540 gaagattggt taattttatt atgttaacat ttgatatgaa taaggtaata gcaattggta 117600 atatcaaagt ggtattcata caaatcatca taaaaagcag agaagaaaag caaacataaa 117660 atctgattcc aaacatataa ctctaagact ctggaggata taaaatctcg gtaaattcat 117720 tagagattct agataattac aaactcattt ctaggtgtta aatgaagagc agaagagtgc 117780 catccagaag gcactactgg atgatgatgt tttccagaaa attaatttcc aagcaagttg 117840 tgacatcaag tgtcaaaact gtatttcttt ttatgcattc ataatatttc ctttaaccaa 117900 aaattaacat aacattgcct ttttgatatt ttaaaatttg tacttatatt tactgtttgt 117960 gcttttttat tgtggtaaaa tatatatacc ctaaaattta ccatgttaac catttttaag 118020 tatacagtac tgtggcatta agtacgttca cattgttttg cagccattcc ataatcttta 118080 tctatttgca ggatgttttc atcttcccta actgaaactc tgtacgcatt aaactctaac 118140 tcctcatcct tcctctcctc ggcccatgac aaccactctt ttactttctg tctctgaatc 118200 tgattactcc agcagtgctt tcataaacag attacattat taatcattat ctcattgccc 118260 taggccattc ttaggaccat tccgtttttg taaagtgtga taatgaagta taggcttctc 118320 caagagcttt ggcaaattta catatttcct ccatattcag ggtctcagct gtgatgtctg 118380 actgctatca cttttgcctg agtctaaggc cttcttctgt ctccgacaca gcttcctcta 118440 agatgcggtg agatctagga aattagccat tatgtttatt agggactgtg gagaaggcaa 118500 gtgagcaggc agtggttgcc taattttgct tcctcaggtt cttcttttta agccactcta 118560 gggtagagcc ctgtgtggag gtatgaggtg gctgacattt acccaggtgt agcattgctg 118620 aaacattcta caaagtcact tgatgaggga tctaatggta aagtttgatg tgagtgtgag 118680 taggagatga aacaccacca gccttttctt tataaaagtc catgtgaggt tgtacagtgt 118740 tttctaggga gtggaaatat gtcagattta aatagcagct gaatgacaag ttcaattgat 118800 tcttgtcctc cactggtaag ctaaacctta aatatcaaag aaaaaaaact gataaaaatt 118860 tttaaatata aggagcaatt aggccgggca cggtggctca tgcctgtaat cccagcacct 118920 tgggaggcca atgtgggcgg atcacggggt caggagatcg agaccatcct ggccaacacg 118980 gtgaaacccc gtctctacta aaaatacaaa aaattagcca ggcgtggtgg cgggtgcctg 119040 tagtcccagc tacttgggag gctgaggcag gagaatggtg tgaaccccct gggaagcgga 119100 gcttgcagta agccaagatg gcgccactgc actccagcct gggctacaga gcgagattct 119160 gtctcaaaat aaataagtaa ataaataaaa taaggagcaa ttaaataaca cttaccaagt 119220 aactcagaca cactcaccgc cttttgctgc tgggttatct tatagtttag gtcactatag 119280 ttaatttcaa tttttacaag attctcccta gggataaaaa agagagttaa tatagcttaa 119340 gattgtcagc attactttta attcccgtcg aataaaggta atattcattt ttgttttata 119400 tgctatttaa tgttaaactg acacagaatt gttaatatta atcacaatta gcaggatgta 119460 gaaggcattt tgtcatgttg gtttttaaga aaaaataatt ttccccaata attttctgca 119520 cctcattcct gagaaactag aaaaagacat taaatgtaaa tatctactga ataaaatatt 119580 tacaaacatc taaatgtgtg tttgattttc attcaaatct tggttctgtc ttttgttact 119640 tacatgacct tcaagaaatc acattggttt ttttaagtca tggtttcctc aagagtagtc 119700 aggattttag agctgagctc acagagttgc agtgaggcaa aattaaatta tgtatatgca 119760 aaatggacac agagcctgac gtatattggg tgttcgatga atactaatta ttaactatgt 119820 gtcattaata tacctaggca aagactctgt tttcttaatg tctgtttgaa atctttatac 119880 atgaccagag tggctaaaca tcttttaaaa tgatactaat agctcgttac acggtcttga 119940 aagccctcac tttctctatc accagcctag acccctacct tgaacttcaa taatatattc 120000 agttcattct tatatttcta cttggatgtc taatagatgt tccaacttta atacgttcca 120060 agtcaaatcc ttcacatttc ccttatttat agtgtaaatt ttcacactcc atagccagtc 120120 catcaggaaa gcccattttt tatgttacct tcaaaatata tctggactcc tatgacctct 120180 caccccattc aaagctacca cctttgccca agccttcatg acctctcacc tggatttttg 120240 caatagcttg ctaactggtc tttctgtttc caactttagt gcaccccact tcaacaaata 120300 ttgtcaggaa ggactttcct gacaataatc cacaaccctg aaaacaaccc tccctgactc 120360 tggcttcccc acactgcttt tctgctgcca ctctctcaca gcacttaatg ccatctgaca 120420 tgactatata cagtggtcac caatttccat cccagtggac aggagtgcag caaggcagga 120480 tttttgtctt ttatacactg aggtatctct aatacctaga cgactcctga atcctaacaa 120540 ataataggtt gtcccttaga tatatgtaaa atggacaaat aagggagcaa attaaagatg 120600 aaaccaccaa atgaatggat caatattacc ctgtcagtgt tataagatga atctgaaacc 120660 aagtacctca tgattttaaa atggcaataa taataatagt attcattaag agtggatact 120720 attagccgag tttcctagaa ttatgtggtg atctttaata cacctaaaga acatggctgt 120780 aaatcactct ccttaatgtt tacaattttt gaaatcaata attccaaata tacttaattc 120840 aaaatatatc attaatatgt ttacttaatt tccattggtt taaccctgag taacaggaga 120900 atgtactatg tttaagtaga taatcaggga ttacagtaat atttacaaat gataaaacag 120960 ttcccaaagt aatggaaaaa taaagttgtc ttggaagtcc agaaatttta gtaatattcc 121020 aatctctttg tcccccattg tgctcctaat ttgcatgtgc actgataata ttgtttaatt 121080 tcccaagcac aacattcata atcatatcca agcattatcc acctgtgatt ttgagagaag 121140 ataaattctt cccctgcaaa aatattgtga tatttgctca acttctctca ggcagttgtt 121200 ttgtctaatc atttgggtct cactgaatct gtgcctcatg gcaatagtga aattgtccag 121260 gatttaacag gatttgagtc caaaaatatt tgtgtcagga gaggcacaat tataaagtaa 121320 aaactaaaaa ctgaattttt ttattgtatt acaaaatagt ttctaaaaaa gcttccagat 121380 gcttgaacag gagtgctgca ttaggggaat gaattaattt ggcataaaga aaacaatttt 121440 atacaatttt tatttcaaaa tttgttaagt taaaacagaa caatttttaa aattcacttg 121500 ttttactaat gaagttggcc taacgtcaga cacaatttac ccttatcttc aagggtaaac 121560 tccacactgc ctgctacagt caagctaagc ctctcaacat tttgaggtac agcagcagcc 121620 ccatgaggcc gtggattgac catgaagtag catttatttc tgtcccagga agacaaaaac 121680 atacacctcc atggttaagt taggggtcgc ttcttttaaa atgactgctg ctaacatttc 121740 acatttgccg tcctgacaaa ctgcacttct gttttctgat ttaccactct ctaatttcca 121800 gtacctttgc acaaaccagt tgcttaatta tatgtgtgac ttctaactgt ctctgtgtgg 121860 gagttgaaca acccctgagt ccagccacct ttgttcagct gctcacctta gtcttcctgg 121920 taatggcctc cactatttaa aaccatgtaa ggtccctaca tgtacattat gtgataggat 121980 taattggata ataattaata attatatgga caggaaacag aaaaatactg ggtagaagag 122040 ggcggttccc tggcaaaggc ccaccctcag acctggatac ccgtggccct aaatgagaac 122100 aggcatttct gtttttgcat ccaaaaagtt gccttttggc ccactacacc ccctatcctg 122160 cctccatatg aaacccaaac cccaagctcc agaagagacg agaagaccaa cagatcaaca 122220 aaccagtgat ggcaaaatga tgtggcagag aaagagagaa gagaaggcac atctgaacac 122280 caaggggagc tcggccgggg gtgttcagag aagaatctag tcacaggctg cctgactcca 122340 cgcaaagatc acaatcccac tccatacccc cttctggctc ctgatccatc tcactgagag 122400 ccacctccac cactcaataa aaccttgcac ccatccttcg agcccgtgtg taatccagtt 122460 cttctgggac actgggaaag accttgggat acagaaggct gtcacactgg ccctctgccc 122520 ttgcgataag gcagagggtc cattgagctg attaaccctc cagccatctg tagacagcaa 122580 agctgaaaga gctttgtaac cctagggttg caggcaccca atgctagaca ctaccatggg 122640 gtaggagccc aaagcgctcc ccctggcctc tgcacctgcc cgtctgcatg ctctccatag 122700 gagtttgagc tgcggggata ccgaacaggc gagcaacacc ctgttgcaca ttttgcaagg 122760 ggaatcagag aattctcccg tttcaagaat aatctgagtt ttggaaaaaa aaaaaaaaag 122820 aaaaaaaaac agcattccct aagatcctct tgtttcatac atggctttct tttctgattc 122880 accagtatgt ctccaaaatc atgcctactt tctctgtata ttaagtaatt atgaaactca 122940 ggagatttct atcaaccaaa agacacagga agtgctcttc taagtcatat ttaaatacat 123000 caggaacaat attaaaagca aatcagtttc ccacattaaa acaaaatata taaatagatg 123060 tctgtgctgg agtcggaagt gtaaacaaac ataattgtaa attttacagc ctgtttatca 123120 ctttatctgc tgtttgaaat cactccatta gccctgtttt gatgaaaatg ctttttcttt 123180 ctgtaaagat cactttgtta cctcagcgtt tacatgggaa ttttttttta ttttattaaa 123240 gtaagcacaa ggctaacatt ggaatctaag catgctttcc aaattatgca cacatctgtg 123300 aaaatgtacg tcaaccttaa gttagttaac atatttttat tttaatagat ataactacaa 123360 ataccaatat actgtatata ttgctgcttc atgtcctagt cctaaaaata caaagaagaa 123420 acaaagagaa atgctaatgt tgcttccctg ttaaaaaacg aagagttatt tatgaagctt 123480 gatttgtaat ttttaagagg ctgtatggta tagcagaaag aggactaatt taattctcag 123540 ttaatcaggg gacgtggaat catgtatctg agttgcaaag acatggggac cagcgtcaaa 123600 gttcttgggc ataacttctt tctgggcccc tgactacctt cacagaacca tttcttaaaa 123660 tacgatgtcc ttcagtttcc tcttctgtaa aattagaata ttaatttctg tttcatctac 123720 cttgtacagt tattagaaga ctcanagtgt ataatgtaca ggtaggaaat tttcatattt 123780 taaggcatcc attattatca gtcaacatta ttattttccc cagtttgggc aataaatgtt 123840 tgatcatgag caagttactc aagcaagcat cattttcctt ttcacacaat tacagtaagg 123900 agttgaaaga tatgatctta tttactttta aaattctatg gtttcattgt cagttcagtg 123960 aaaaaagcca ttgcctccct tgattgatta gaatttcatt tcctgagtct ttgagggttt 124020 tcaataaaga aaaaaaaatc tatgttaatt atcaataatt taaaaggcta gtaaggttac 124080 ctgatgtatt tccggctttg attcaacttc ttggaaagat atttcaaagc tttttgactt 124140 ggaaaagagg aataagaaat agtggccggg tattctattt cttcacaaga aacggggcag 124200 ctagagttat gtgttcctac tgtacataaa tccttaaatt caatgtggtc tgaaatgaaa 124260 atcaggacag ggaattcaga gaagagaaat atttcatcat gggtaggata gttttataaa 124320 gtttatcatt tcactttcaa tattaaaaaa aggtggttac caacaacaaa tggtgtttac 124380 tctgaaacaa atatgttact ctgaatatag cagaggagat acaatataag gcctggaagt 124440 tccaatcttt gaattcattg agtcactgga ttgggatctt tattggattt aagaaattat 124500 tttcaggaag tatctgggtc tatttctgta aaatatcaga tatttggaca gaagaatatc 124560 tgggtctatt tctgtaaaat atcagatatt tggacagaag aatatctggg tctatttctg 124620 taaaatatca gatatttgga cagaagaaag atggaaagtg tgtaaacatc ttcagatttg 124680 tctcaacata caatggtaag aaatgtgctc ctgagtattt gcagttcctt atttctgttg 124740 gcccagtcac ttccaaatca accactgagg ttctctgttc agtgatctct cttggctcct 124800 tcagagcaac caggctgtcc atgcaaatgc atactagaaa aaagtccctg tagcaggtag 124860 tgtttgtctc cagcttccct ttggaccacc tcccagtgct cattctcact gctcctcatt 124920 catggtagat actcaggttc ttgtttttcc tttggttttg atactgaaac cctttgttct 124980 cgtgaatcag ttgtttacca agacctcctg gttactgacc aactttaggt ttgcttttca 125040 gactctaact ttgacttctc taggagaagt caaagcatgc atctgctagt tctttagcat 125100 tgctggcaaa gaccaccact cttctcctac actcacatgc actcatacac tcccatacac 125160 acataggctc acatgcacat actctcacac acacacttac atacatatac tctcatgtac 125220 ataatactct ctcacacaca caaacacaca cactcttctg acctccttat cccacaacag 125280 tccccgctaa actgcatgca tggttttttt ccccaagctc tccctccagc ctccaacaag 125340 gataagaaat ctgaagaaat ctggcaacat catttcaagt ctattggtta gtaatacggc 125400 ttttgtatat tcagcctgtg atctacataa aaaaagattc cttctaccta tagccatacc 125460 ttgaaatatt ttacaaccat tgaaaagaac cttttaaatc tattcctgct accctggaga 125520 tatttccaca atgggttaga aaagcaagtt gcatagaagg gtatatattg ggattctgtt 125580 ttaataaaac aaccaatgac ttcacgtgat aaatatggag atgtatatgt ggagaaagtt 125640 acagtaggat atactaaagt gttagcgatt attgcaggtt aaggtaagag gtgtgaaagc 125700 aaaaaaaaga gggggctctt taataagaac ttgtccccaa taaatatggc atgtacagtt 125760 ataattccat ttatgaaaaa attttatatg ccaagtgcat gcaataaagt gaatgaatga 125820 aggactattc ccaatcagtg agaggagttc tcagtaataa aacttctatt tatttttaca 125880 tgatttcctt tctattctgt agtattttaa ctattcacaa taaacttgtt ttatttgtga 125940 aacaataaaa agcagctagg atattttgat tataaaaaag aacaaaattt aaaaatttag 126000 atgtgtctta atatcagagg gtttttgttt gtgtatactg cctgtgtttg ttagctacta 126060 tttaagaaca cttacaaaaa gatcttctct tatatactat attctgttta atttctgggc 126120 tatccatagt ttgacttaat gtcttatatt actttgagca actttaatat aaatcctgaa 126180 aataaaagta aattaagcac ttaccaagta caggagaaac acagctgaag tacttttgta 126240 ggtcacattc tatcccatat ccttaaaaat aataagtgta acatatagag actttacatt 126300 ttgtaaaata agtgatctaa tgacaaaaat acctttctta cctacatcta tccatccatt 126360 catccaccca cccattcttc tatatgaaca tctattcttt cataagcaaa actatggaat 126420 caagttagta tccagtgaaa atatgtgttg ggaggaagaa taatgctttt gtttctttcc 126480 atacctcttt ccatctgtct atatcactct aaaatcttta agcgggaata ataattttca 126540 aatgtgagta gttttcaaat ttaaaattac tttcacagtt atcatctcat ttgatatggt 126600 tcacaatcct gtcatatgag aattttcatt ttacagataa agaaactaag cctcagaaag 126660 agtggcttgc tcaagaccac acagttaggg gaaacttggc acattctgta catcttccat 126720 cctgctccct ctatccattt acacacacac acacacacac acacacacac acacacgttc 126780 ctgacagact gccattccag aagtaggatg gggagggggc aggtaagtac caatatttgt 126840 taagtaccca ctatagattg tgagataagt agaaaatcac tatgtatcag agctgggttt 126900 actcctacgg ctgattctaa agacttagtc ttttagcctt aatacaatat ttgtctattg 126960 ctatagccca tgtgactcag cagttagcca caatgtttag tcagtaatct tgaaaataac 127020 caaaggagat tgtccactta caacgtgcgg acaatttgtg gaatgtttgg ctaactgtaa 127080 ggaagtgctc tttatgaata aaatacaata agtaaaaaaa aaaaactacc tccaacactg 127140 agttaggaaa aaacctcatt tttctgaatt atttcttgct ctggcaataa tatttcttcc 127200 attataaaga gcaattcaga aaaaaaagat ggtagatgga acacagaaaa caaaaataaa 127260 aacaaacaaa caaaagaaca atgaaatttt ggaaataacc aatatctgaa aagctgtttt 127320 ggagattgct tcctcaaatt gttagtggaa ttacctcagc gaatttgttt aacgtttaga 127380 atcttccgtt tattcatata tcagtacact taaagggatg tgtgagaatc taaccaaata 127440 agatcatatt cctgaatgca tttgatatat atgctaaagt tcatgcagat atcattcttt 127500 gtgttttata tagcaccttg cctagtttct ggtccacatt agccagtagg ttttggttta 127560 ataatcataa cttttaaaat tgtcaacatt attctgcatg aaacaattca gacttctttc 127620 cagcaacttt gccaataaat taggaatgag aaatcagagc cagagccagc agttttatat 127680 agaacagcag tttttgcagt ggccagagat caataatctg gatactaaaa agaaggatgt 127740 gatgattatg gaaacctctg gaaaataatg aaaccacttt tgcaaaattt atatcttttt 127800 tacttttatt attttttata ttatatataa tataatatat attttttata ttttatatat 127860 attttatatt tatacatatt ttatatttat attttatata ttttttactt ttattattac 127920 attgaaagag atttgaccta actgactcca tcttgtttct aacctccaag ctgtccttgt 127980 tcattcttgg gcgttcgctg aactaacttt gggaggaact tagtttatgg tttagctctg 128040 aaacaaagac aataacagcc ctttcccaaa ataaactccc ttcctgcctg gggactagat 128100 tgcctatata gggctaacaa attagccaca agattagaaa ttatgtttag gggtcatgca 128160 gctggagggt gcaagattct aaaccgcccc aaattgctta tagtgataac attactattg 128220 taaaagctaa gatcagtgct tgagatatgt tgcagaccct gcctccaatg atcagctggc 128280 accacccaga acaaaaatct ggcccatctg gttttgtgac ccctacccag gaattgactc 128340 agcgcaaaaa gacagcttca actccacatg atttcatctc tgatctcacc aatcagaacc 128400 cctgattcac tggttcactg gtcccctacc caccaaatta tccttaaaaa ctcttatctc 128460 cgaatactca gggagactga tttgagtcat aataaaactc caatctcccg cacagccagc 128520 tctgcatgaa ttagtttttc tctattgcaa ttcctttgtc ttgataaatc agctctgtct 128580 aggcagcagg catggtgagc ccgttgggca gttacaatat tttggcaggt agattgtgtt 128640 acaatatatc tttctagata tgctgtggga gggggatgta agagacctga atacaaatat 128700 ctgacattta catagactat attctgtgcc aggcactatt tgaaccactt acatacccct 128760 ttcattctta caacaaccct ataagatggg attattatcc cctcctccat ttgacagcag 128820 aggagagagg ttaggtcact tgttcaagat cacctagggg agaaattgta gagccgacat 128880 gagcagcctg gttgagatcc agctcttatt caccctgctc cattgcttcc ttgaatgctt 128940 gcctctcacc catggttctg gaaaagttgc aaattgtatg aaaaggaggc ctgaagttaa 129000 ttttacagtc ccaaagaaaa taacttttca acagtgaaat cttcaggctt ggtaagacat 129060 atccatgatc taatttttct ttcaaaatta agaaaatctt ggtgtgctgt tttagatatt 129120 tgagaccatc aaattttagc ctttgggagt aagtattggg gccccaccct gatatcgcaa 129180 ttttctcttc cagaaatctc tgcgatgatt ttataatgta aaatttgtca gtcgaagttg 129240 tacctggcaa atggataatt ttccagtgta gaatttcaga agaatggaaa acaatttagt 129300 gatatgttga tttgctatgt ttcaaattaa gtaaatataa acataaaatt caaagaaatt 129360 taggactaaa cttctggtga ctttgttatt agttgtcatt ttagtctgca aagagaagga 129420 acaggcttaa gaatataata atttttctgt gaatgggtca gcaacatctg taattacact 129480 taataatact taatattcca tttgtatttc aatactctat ttctcccaga ccttttgtaa 129540 ccctctaaga aacacttaac tgtctagtta agtaaggggg cagttagtct ttatttacca 129600 ggaagaagaa aaggcacaca tccacattgc ttttttatgt gctgggcttt gcattccttc 129660 aagcaaccag aagtgctgta gctgctaaaa ttctgcagct tgatgttagg attgcattct 129720 ccccaagggt attcttgatg aactgtctta agataagata aatactgggt tcaggagatg 129780 tgtttacatc aattcactta ttttccatca agaagctggt acacattttt attttcctct 129840 cagagctctt tgattttcaa aaccaaaatt ggttacattt gtctattttg cctactctat 129900 tgacctgaaa ttccctgaag ctaagtataa tagtgatcct taaacatata tcccaaatgt 129960 gaattaataa agtcagacac attgagtatt ctaaaaatca actcatataa ggaaaaaata 130020 tcatttatgt aaatgttata ttagtgtagt aatcagctca cctacaaaac agtgaagaaa 130080 atcacatttt aaaaagatcc tttaataagc aactattatg tgccaggcaa catggtgttg 130140 cagaactttc tccttagttc agctaaaact gggctcttgt cacatgacca ggaaaaatga 130200 ggctcgcggg caaatagaag ggtgaggaaa atggaattta ttgggtgaaa agaaaaaaag 130260 aaaaatgact ctcagctaag tgagagagag tcctgctggt aggtttccca cctcacagat 130320 tgaatctcag gccaccacac aggaacatga gaggccaggc tcctccccac tgcaaacagt 130380 gcaaacttcc caaggctcca ccccttcctc ccaatgtgca ggtgggcatt tttcagaaag 130440 aatcaggtgg gaaaggacag ccttcatctg ggacaagcag tccagttttt cagccttcag 130500 gctgttttag gtttgaaggc agggtttcac caggggccct tggctgtctc ctgtgtctat 130560 caatggtaag tgctttcttt atggtacttc acttaaactt ccttactatg aaagaaaaat 130620 attacccctt ccctatttgt gcagagagga gatgcaaatt caggaagcta attaaagaag 130680 ttaccgggca agtaagtggc tggcaaacag aaacctacca gaccagcctg actccaagat 130740 tcctgctttt cccactaatt ttctccttcc cagatagtct cattgaagaa tcatagctca 130800 gcaacttagg tacctcttta tacttaatga ccctttccat ttctaatgaa atgtaaagtt 130860 tggatttgat aggcttccca gctttcactt acatagtttc tgtttccact aatcatgtac 130920 cttcagataa aggaaaataa tctgaaatga aattttggaa tttctaatgg tgttttacag 130980 gcatgggaga aaggaatttc tagtgaaaag cattacttat gtgtttgatg cattcactac 131040 cccacctaat ttcctcagac tcgagtattt accttcactt ggcggatggt tacccttgcg 131100 tgcattccca caggtgacaa caagcctaac ccatcaaact gtggcacctt ctttggtgaa 131160 tggataacaa agatgatccc agcatcaacg aaaccaaggg ctgggttatc agtgaatgcc 131220 tcctgaaaca aaaatatgtt tttgcccaaa ttgcaaattg actatatcag ttctaaacaa 131280 gtttaggatt tagttttatc atctcatgat agcgtttgac taatcctaaa tattttatgt 131340 tatatttcta tctctttttg tctaaagtgt atgaaaatta aagacatact ttagtggttg 131400 aatcggtgaa aggaaactat cttgggtccc ttcaaggtgg ggtctactca ggacagtcct 131460 gaaataattg aggcttgtct catcattttt cttgattgat gtctggtaac cacaaaggga 131520 tcctgagtga agctgacctg gcctgcagca gctagcctat cagtgcttgg taccagcttg 131580 ggcaccttat agcccaaacc aataggacga ttgctgaact ccaggaactc tttcctccag 131640 ggatccctga tcttccattg tttttcattt gggggtctga ggttcattcg ctattaaaaa 131700 aacaaaaaac aaacaaacaa aaaaaactcc tttttttgtg ggagtttcca ctgcatccac 131760 caaggaatgt gaacctacct gcttctgcat cggcagagag cagttttcag cttgggcccc 131820 atcactaggt aagaaaactg gtttgggatt ctttcttgca aattcttttt aaagaactaa 131880 agttagcatt aacaaccagc tgatgttaat ttctgcttac acttagagcg ctcagaaatc 131940 atataatttg tgtgatcact gttagttttg cttaactgtt ttgttgtttg tttctctctt 132000 gtggggttgt gtgtgtgtgt gttttggttc tttctctcat tggatttgac caactcagaa 132060 ccctctagct catgagtata gaattttcca ctccaaagaa ataaagcacc ttgctcccct 132120 aagccttttg gggcattctc atgtgactga gaatcacatg ggggtgtctg ggaggaatgc 132180 tccctaaaat gtgcagtggc tctaaataag tatccccctc agaagaatat acttagggtc 132240 taatctcagc tggcaggtgc atgttaggag ccaacccctg ctgcatcttg agcacctaac 132300 acactgtgcc aggtagctgc aacacaggac gaccaatctt gttcagggat aacagccctg 132360 aaaagctaag tctgctagca gcacattttg ggtccaacac gtgtcccaac ttggtcaaat 132420 ccaaagggga actctaaact atggggaaca aggcctctga agtggaaaga aaacagcaat 132480 caagaggaaa aaaaaaggaa agatttttta ttttgactac taaaggggct ttatttacat 132540 aacaaagcca cctttttatc agccagacca aactgaaaga gcaatggctg cacttctgaa 132600 atatggtaat gaggtctaaa aagaattttt ttaaagaagc tcagtgtttc aaagtcaact 132660 taattaaaag attaacatcc aagatgtgtg tgtgtatgtg tgcatgtgtg catgtttgta 132720 tttaaaaggc cttcaggttt ttgtgggttt tttttttgtt tttctctcct aagactttgt 132780 cttttttttg agcaaaagtt ttttttttcc ttcagttgac tgaattccgt tttcacctga 132840 tcttttgact aaaatagtta ttgcaacaga ggctaatctt gggtttttaa ggaagagtgt 132900 agttaagaca ctcagaaata tctttgttaa aaaaaaattt aagtgcactc tgaaagcatc 132960 acagggtcta acctcaaaat aattctaccg ttttttggag acccaggatt caatgtgagc 133020 tctgcccaga gcttagagat ccagttaaaa tataggtagt ccctatctaa ataagattgg 133080 tctccttata caatattatg atagagttct ataattttat gttagatttg gctcaaagaa 133140 aaataaaagc atctccctct agcaccaaca gactttttct ctctgtacct tatgatataa 133200 agtttgctat tttattttca cctgagttgt ttcctataat aagcaaattt aaggctattt 133260 agctaacaac tgcctagggt tgtaaaacag gttatcaaga atctgaatgt ctaagatagg 133320 aaaaaaataa taaaagggtc tttatgaatc tataaaatgt acctttattg gcatacctaa 133380 tatgtctatg tatttatatg tcatatacac aatatttcac tacagaaaat atataaaagg 133440 gctctaatta attggcttaa agaaaaataa aagtgtttaa atcatatatt ttatcaggaa 133500 aaaagaaaag acagttcaaa ttctttttca agtttatgta acttaagtaa aatctttaat 133560 agaaaagcta gctttaaaat tactagtaaa gtaatatcag aaatgtctta agaattgcca 133620 gcatactttt tttgtttatg tttattaatc aggctatttc aacttatccc tgccaaacac 133680 tataaaatgt caaaatttgg catagagatt acaaaactgt aaacccagcc ccaaacagaa 133740 tgatcattac ttgtgtagtt tttaataaat aagacattga tattggttta atgaaaatag 133800 ctgcatctta aattttcaaa attaccataa tttctaatct tgtggcttta ggcagcctag 133860 tccacaggca gtaaggaggt ttgtttggga aaggactgct attgtctttg tttcaaacct 133920 aaactataaa ctcagttcct cccaaagtcc aggaatgaac aaggacagct tggaggttag 133980 aagcaagatg gagtcaatta ggtcgtatct ttttcactgc ctcagtttta tttttgcaat 134040 ggcagtttca taactttaaa tcatgactat cgtagttttc ctaaataatc taggtgaaca 134100 attaaaataa aatagttagg taagggataa atacttgtag acaaacatgt cgtaacttag 134160 aatataaagt tatattcagt taaataatag atatttcatt atgtgggtat tttccaataa 134220 atatatatta tagaaaaaca ttcttgctaa aaaaaagtgt gtcctttata aaaaaacata 134280 aacaaatttt gtctaattca aagcttatct aaaggttatg tataaaacaa ggtaagaaga 134340 acaagcaaac aaaaagagat gtaaagaaag ctataaaaat aaggaggttt ttttgtggta 134400 agacagctta aagagaaata atatggtaaa tttagtccta aaataaaatg actggttgtt 134460 taagaaagga gaagtgttca ggtcaaacca gaaagttcaa gcatgtcatt aatagtcagt 134520 gtaagtcaca ataaggattt attttttaaa aaaccaaaaa ctttaatatg atcaagttgt 134580 cacattatta ttaagtgttg gtttgcttag gaaaaaaact gagataaaaa tttttgtttt 134640 caaattaagg ttattacatc catgtatctt cctgtatgtg cttttaaagt ccttgtgaca 134700 ttaagttaca gggctttgac tccagggtct aaaaaggata ccaagtccta ctaaatctta 134760 aacactaaca gcaattaaat cctcatcttc aggccccaca gcagattcca ataaaaataa 134820 aatgcattcc tggccaggca caggaattca cacctgtaat cccagcattt tgggaggctg 134880 agcaggtgga tcacctgagg tcaggagttc cagaccagcc tggccaatat ggtgaaaccc 134940 catctctact aaaaatacaa aaaatcagct gggcatggtg gcgcatgcct gtagtcccag 135000 ctacctggga ggctgaggta tgagaatcac ttgaacccag tcagcggaag ttgcagtgag 135060 ccaagatcat gtcactgcac tccagcttag gtgacagagt gagactctgt ctcagtaaaa 135120 aaaataaata aataaataaa atgcattttt gagatgtggg gccagaaatt aaagccattc 135180 aactcctcga ggcctaggga ctattgagga agaggtgggc atgtgagatt gcaatgggcg 135240 atattaaaag acaaaataag ttcagtttct ctataaatta atcacgactg tcaaaggcac 135300 aatgatgcaa gaccagcata tggactcctg tgtcagatta acaaggtttt cttgaagcat 135360 taactaactc cttaataaag atcataaagg ttataaaagg cttatggaag ttatatttta 135420 tggtcaagat taaattttat agattgttta caaaattttg gaaaacaaat ttaattggct 135480 tcatgctgtt tttattaggg cttcttattt ggaaaattaa gtctcctctc tcaaagaatg 135540 aagttttttt ctttttttaa aaaaaaatcc ttgagttatc actttggtta aatgaatgac 135600 tttacaataa cctgtaatcc tatttcataa tatcaagtat tttacacctt tgatatttga 135660 agatctttct aaaatcaaat tataaattat gtctttttct gacctaatta atcctttaag 135720 atattagttt ccctaaagtc caaaaatgac ataatttggc ttacttggta taaaattata 135780 caggaagcat tgtcaaatat gaaatggtgt ttggttttat ttgggctgta tttatgtaaa 135840 tgttattggt aagtgttcca gaattaatgg aaaggcctgt aattctgata tgacttagtg 135900 tacattatca ataataataa taattgttat gttaaaatta ttgtgtacca ctgaggtaac 135960 aaatttcctt gtcaattgtg tctttgacta tgtctgccct aaaacctttt ttcatccaag 136020 gacaattgtg ctcatgtttt ggtcctcttt agaaggtgtt tttataatca gctacaaaac 136080 tctaacaggt gcccttaaat gcaggtttct gataactttg gagattgtaa catcagaaaa 136140 gaggaaaaac tttcaggact catggagagc taaaatgttc atgagtatta aacagaacag 136200 gaattaactg catggactca aataatcttt tttacttttt acttaaaatg tttgctgatc 136260 ctttgttttg tttttcagag tcttaaaact tttattttga gctacaattt agaatactcc 136320 tatgaacaaa acgtggagca tactttatcc tgtctgcctg atttctccag aatttggaaa 136380 ctatttgtgg atattcttaa cttgtggcaa tacagttatt tgcataagtg caataagaat 136440 ctgttttcac ttgtaacagg acacaattgg agaaactggt tattttacca aggcttttac 136500 tggaatggtg tgctttcctt taaggaatca aacttggctt atgaaaccaa taatgtcctt 136560 ggaaaaactg acctcatatt ttgtgtacag agtccctgta cagggtttct gacctgtggt 136620 aagtaaagaa tgtcactttc tgacaggccc agaagctcca agtttatctt ggaacctcga 136680 gtggcgagga gattcaccca actcataggt acttgatggc acaaatctac ggctgggctc 136740 ggcttttaaa aagtcttatc tgacattcct tctatggaac aaagttccac caaaggcaat 136800 ttaaaagcct atgtaaaaaa taactattct tggtgcactg tatacaaata atttggcaaa 136860 gtaaaataaa gcaaactagt cctaacatga tttgtcttta gcaaaaatgg gaaattttat 136920 gtcctaatta atcctttagt taggattaga gaagagagaa aaattatgtt tccaaaacta 136980 gggtacacct gttgttagat tctagtcttg cccagtgttt ttcaattttt attattttct 137040 acagtttgga ccaaattcta ttttttcttg gctacaagcc ttcaaaataa tgttttcaat 137100 ttttttcttt tttttccccc atatttccta atttggagtc actgaaaact aagctgtgct 137160 ttcttaaagt ccggcaaact gaagccagtc aacttaaact ttagaagaaa gtaactgcag 137220 cctatttaca tacataagcc acttttcata cctgcctact gatgtacgga cttcaaagta 137280 acatggccta tatgaatatt tccagtattg ttcttttttt tgctgttgtt tttctccctt 137340 cctcccacta ttttctcttc atagaacatg agactttgca atctgctaaa aataagcttt 137400 tgggacctac ccatctagta ataaaccatc ctaaccatga gaaatcagat gaaaactgag 137460 accagagact catattctcc taaaatgctt tctcctaaag attttttttt taaagggagc 137520 aggggaatgg gaaaggaaat tatcttgggc tctgtcaaac tgggagctgc ctcccattct 137580 atttaaagtt attcctttgc tcactgagat gaatgcctat tctgattgcc tcctttggaa 137640 aggtcaatca gaaactcaaa aaaaatgcaa ccatttgtct ctcacctacc tatgaccttg 137700 aagcctcctc cctgcttcaa gttgtcccca cctttctgga taaaaccaat gtatgtctta 137760 gatatattaa ttgatgtctc atgtcttcct aaaatgtata aaaccaagtt gtgccctgac 137820 taccccggac tacctcagga cttcctgagg ctgtgtcatg ggtgcctgtt cttaactttg 137880 gcaaataaac tttctaaaat gattgagact tgtctcaccg tttttctcaa ttgacattca 137940 ttgcttaaga cctctatagt tttccatgga tcacatttca ccttagatag gtgcaggtaa 138000 gtatttacaa ttactgttaa tgatcttgtt ccctctggcc ttctcttcac ctagttgcct 138060 tttagttgct tccctcttgg ctgttttctt tgactcattt gcttagaaaa tcatttggcc 138120 ctcttcattt tgcaattaaa atccctgccc ttatgttaaa ttcaaattca ttttcccata 138180 agagtgaaga tttacactat ttctttaaca ctaatgttct ttttaataaa tcttttgtca 138240 gatgaagaaa tttaagagta aacttctggt ggctgtcatt agttgtaatt ttaggctata 138300 agaagaaggc acagtcttaa gaatataaat ataaagaata tcgtctatga atggatcaac 138360 tgcctatgta attacattta acaaacatac taaatattct atttgtaatt caaaactcta 138420 tttctcctgg cccttttgta accctctaag aaacacttaa ctgtctagtc tggagaaata 138480 tctaaatgtt atttacctat ggaacattgc attgaatttg tagatgttca ttgcattcta 138540 aaaatatttg gaaaattcta gtttgtaaaa attgataata taatgttcga actgaccata 138600 gggctaatat ttgaatgact cgaaagtatt aatcaaatac attacaagta tatgtaggta 138660 catgcattaa agtttgcagt gttatgtttg gacgtaaaga aaaatatctt gaatgggata 138720 caaaaggaag ctatgaagag tacattactg gaaattcttt aaaagtagcc aatggcatga 138780 ttcacttgga ttgggaggtg aatgcaggaa catcaatttt acttttcatc tttgtcatta 138840 attgtatgat tttggatact taatccatca gcattttaca atcctctttt ataaaaccat 138900 aataataaag tagaatgcct taaaatgtgt aatactagaa gtagttattt aaacaaaagg 138960 tcagaattaa ctaatcttgg gtgggagcta tactaggaac ccttgaagaa aacaccactg 139020 catttcttag gtgtttgaac atctgcctcc caggactaaa cacttagaac ttattaggtc 139080 aatttcagct ttccaaaagc tgggcttgga caaaatctat ttatataata ttctttatat 139140 aaccattatg ttttccatca cttttccaga tatgcattct atagtttgtc aaatgtgtat 139200 gtagagtttc tttctaaaac gtgatattta cataaacgta tttctaaaat tattgttcaa 139260 tgcaaagagt tctcttaaaa tagctacata cattgcaact tagaatctct gggttttctt 139320 attttgtggc atgaacattc cctggagtca ataatttaag tcagtaggat agctaaaaat 139380 ctccagttta tgcctcaaaa ttttgcctgt ttaatttcta agccagtagt tatataaaat 139440 ccataggagt ctagactaaa ttaacaaaaa ctgaaataat atccaatgtg tgggaactat 139500 ctgatatggc ccaatagtct gatcaagttt ttgaaacccc cacacattct cgtttgtctt 139560 gaacctggag aaaattcagt acctgattca cattgaagag taagctcaaa cctcttccag 139620 agacactcac ttttctcttt gcttggagag tttcaccatg attaaaagta aaacaatttc 139680 catattcagt gaagacatgt gcaaaatcct ccaatatgta aatgaaccaa aaaaaaccat 139740 caatttgata tttttgtcac ttctcaagtt tgccataacc ttttttttta aagcacaaat 139800 acctaacaag actgcagaaa tatcagagag aatagtctga tttatatgtg tattcagtga 139860 tagttttttt tttttggtaa tatgcattcg tcaatttgca tgtgattatt taaaggagac 139920 attttcatat aaaattaagc atgtatttaa aacatttaaa aaaattactt gtttaatgtt 139980 ttcctgaaga tgtatggaaa catgttttta tctgtggagc actacttcag catcctttct 140040 tggaggagct atgcctaatg taacctgtat ggtcacagtg tggcagtcaa tcacatgacc 140100 cttttactct ccttttaccc tccttttcca accactacag aggtgtgcat gggacacaag 140160 ctgggccaag cttaaacctc accttcttgc ctcagtggtt ggttcaagaa ttgatatgtg 140220 tcccaagtgg agctaattag agtctttggt gagattatta agtggatact ggagtgctaa 140280 actctgataa tgagtctagg gggagtcagc agccattttg gtgaggtgtg gggagaactg 140340 atacagaaag aagccaacca gaattatgag atggaaacaa agtcagaatc ttaaagacag 140400 tcttggagtc cgttggttca gcattttttg aagccagacc tactgttgga gttcctgctt 140460 aattaagcca attcattact tttagtcttc agtttactag aacagaaatg ggtttctgta 140520 atatgcaact gaaaaaattc tctaacatga aattttttgt tggtaagtta aacttgttgg 140580 tgaataaaaa cagcataaga aagcttactt tccaagttaa gagtttaggc cagaacctca 140640 cctctgtgaa atgtctctca ggcatttttt tctgcttact gtgtgctaag gcggcctacc 140700 aaaaacataa gaaattgtat tgaagaacaa agaatttgtg tttacttagg ttcccctaac 140760 aacacaggct acagaattgc tgatacggga aatacggcat caaattgcct gtatagttct 140820 ttattaatag attacagata gatctagtac tttatcttaa atgtagatta attttataaa 140880 caataataac aggattttgt atttgttatg tatttgtttt gtatttgtta tgagtgaatg 140940 tttattatat ataatatata ctagttatta tatataatat ataatatata ctagttatta 141000 tatataatat ataatatata ataataatat ataaattttt catagttaaa tgctgggatc 141060 ctcttatttt ctatagtctc taatgcaata ccataagcat atatgcagag tgatggatat 141120 ttggtgatta taaaaatact atgttggaat aggttgtttg ttgatgaatg aaaaaaatta 141180 aaatatcctc ttgtgttaaa ttgtttttct tttggaagga ctatactaca agaaaggcaa 141240 tcaagttttc tcttttctat ctttccttat ctaactgctt gaaagaggaa atttgtctca 141300 tgtgtattcc atacccctga tatcataagg ctgagctaat gaaatagaat ctgtaagcaa 141360 atttggattg attcaccatg ggagtaaatg ccgtgggagc tcaatgtgaa acagtgcagt 141420 aacggaactt attactttga ttatatttga agaatagaaa atcgttacct ttgggctaca 141480 tggctttcca aaaaactcac agtccaacaa agtgctattg ttgagataaa aacctttgtt 141540 cctgataaat tccacaatgc tgaagttttg gtgacttgca gcaaaatcag tagcctctct 141600 agagccagtg gaattggcag taatttcttg aagatggagg actttggata caatgtgcca 141660 taagaaaaaa ataacaccaa atttggctac agcatctgtt tggaacctat tagcaggaat 141720 gaaggcaata gttagaataa tgaaaactta taattatctg gaagacagat accaacatct 141780 agattctatt agcttcttct tatctcccac actctccctt atttgcaaga catttaagca 141840 aaatttgatg cctaagaagt ctttgaccca ttgtgcaatt aatcttgtat ttgcacatgt 141900 agtttcattt acctagaata ttccccagac cttgatctgg atgacaccta atggatcttc 141960 aagattcaat ttaaaggaag aggagggaat atatagttgt taaaaatgca tttaggagcc 142020 agatttcctg tgtgtggcct tgtcctgtgt cagcttagct aaattagaac tgcatttccc 142080 agcattccca tccttgtata tatgtgagtt tcactgtcgt gcaggagctg tggtagctca 142140 cccacactgc aatccatctg ctgactcaac ttgctagtat cgggcagcag ccaggaccac 142200 agctcctcca gctcctttgt gaccttttcc ttcagcttct tcaaatactg ggttaggtaa 142260 gagtgcgacc tggtaaagat gacagctatc ctgcaggtca ttcacaccat caagattgga 142320 ggtagtgaaa aagagaccta cattttgagc ttgtatttgt ttccccgact tcatatccat 142380 tttcccttcc tgctggccct gcttacttca gccccagcac aaagaacagg cgaaagactt 142440 acaaagactg cttaaccagc tcctgcaacg aagtggggct taatccctag aaaaaaagcc 142500 ctttattctg tcattcatgg tgattctgtt tctccagtta ggttctaatt gttacagcca 142560 cttgatggta tgacttagca aatttttttt tcatttccct ctgacttaag tttcctctct 142620 tgtaaaatct gtataaccta cctcaaagtg ttcttttaat gattacatta attgtttttc 142680 ttaaaatttt tagacacctg cctggaatat agtgagtgtt aggtaagtac ttattaaata 142740 aatgattcat tctggccacc aaagcagaag taatctattt aacttaacga ctgtatcact 142800 ttctaattct cttatcttgc cgggcaattt tgttctgcat tccatatggt tcttcatttg 142860 catgccttcc attcctttac aaacatgatg aggactttca tattttagat attataaatt 142920 ttgaaactga acaaatgttc tctctttaaa ttgactgcta aaggcataga acggaatgca 142980 tattccactc ttggtaagta tatggtgtat gtggtttgta tgtcatacct gaacctcctt 143040 tgtgtcccag attttatttc tattttgctt tatattatat tatcttattt aatatataaa 143100 tatatatcta tatgtgtgtg tatatatata catatatata aagagagaag aaaataaaaa 143160 tatttactct aaaagacatt ttttgacata ttttgaaatg gctgctgcag ggtcagctaa 143220 cggaagtggc cttgcaaagc tgtcttttat gtggaaaatt tgcatctgta gaaaacgtcc 143280 attaatgcag ccgggcctcc tcttgctagg catttgctag gtataggaga gattgagatt 143340 ctgacacctt taaaagtcta aaaataaaca tttgccatct tgtctctctg aagaagtctt 143400 catctatata acaaggtcac ctttgctagt caagcctctt cctttctccc tactataacc 143460 tgtataacct gtcttggtgt taaaacctgc tttcagtaat gttctgagcc cacattcttt 143520 ctataatctc aagatgataa ataagcctct gtatccggtt gggatgttgg gctttattct 143580 gaaggctcat gtgtatacac attaaatatc attgtaagcc ttttcttcta ttaataaatc 143640 tgcctcatgt cactaatttt tcagcctatc tttagggggc caacacccat ggcccccaca 143700 atgtatacat ataatttttc tttcaaattc aagccatatt aaatcctttc tcagtcaagg 143760 tagggcatac acaaaacaca aaaatggaga aacaacctca agccaacatt acatactctt 143820 taagagatga aaaactgttt tcacagtcaa taactaatat tgtagcttgc ttaactagct 143880 ctgatacttg caaagctagc ccaggtccta aaactctgta aatatcctcc tcggacttcc 143940 tgattccaag gcactattgg tcatctgtca tggtggtatt ctgtctaact acagtacatc 144000 taataacctt gcatttcttg gtcaaaagta tttcttggat cattaaggaa ttaagagttt 144060 acatcaggaa cagattttgg cagaagtaca gtgtaaaccc tatattgaga gatggacctc 144120 caagctactt caggtttcca actcccagtg acagacaggt tcccaacaat ctgctccaaa 144180 accctacaac tgtgaaatcc tgcacagtca gagccagtat tccctgaata tattcattgg 144240 attatattca aaatgattat gcagagaaat aaaggaatta ctatacatat gggagcaatg 144300 aatacaatct acaaaaaagt taaataggag ggcatcttaa aatattttta agcttttttt 144360 attatactaa taatatttga gaccatgaga gtaatttata tagattaatt aaatttaata 144420 aataaagcct aacaatatta agccaaggaa gaaatttttt gtgaggannn nnnnnnnnnn 144480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 144540 nnnnnnnnnn nnnnnnnnnn nnnnnnntga gactgttctc acaacaaaat atgcacatct 144600 taaaatacca tctggcttct caggtccact tgagactggt gacctcactt ttgaccttgc 144660 cctaaaagtc aactggagtc catatacctc aggtacaaac agctttaata agctgccact 144720 tcctgttcct ctcatcttct ctctatggca ggactcagag cattaactaa cctaagggcc 144780 tggtagaatg taagctggag gtagagtagt taaagtctgt gcattttgta ctggtcattt 144840 tccagttcgt ctgttgctaa gaaacagcaa cagaggcccg ctctagaggg ttacaaactc 144900 cctggaatta tggctcccgg acatggtgta gggaaaggtg tcccacaggg atcaagccta 144960 gaatatcgct ggatcttgta cagtttagta aagggcagga ctggatttaa cataacgtcc 145020 accaaactac attactgaaa atttttggtg cttcactcaa ctagtctcat tgactagaag 145080 tgaaaatatg tttttctcaa tattaccagc tttaaatttg ccataaactg ttgtgggacc 145140 aagccacaga ggaaatgttt ttccgctaga caaagataag catgaggatg ccagcaacag 145200 aggatattcc ttttaaggtc tgtccattgc aaagtacaca cgtacacgct cacacaacaa 145260 acacgccctg ttgccttttg cttgtgttaa ccactgaggg cagatctctg aataataata 145320 catttagtgg gtccacaaga gctgcatttc ctctgctcca gaattcatgc aaggagcacg 145380 gctggggctt ctccactgtc attgttatgg cctcaaatga aggccttgtc accaggcggg 145440 ggcgggaagg cactggagcg cagcagcatt gagagcatcc gaattccctc ctctgtccct 145500 gattggttga agaactgggg agcaccgacc agccccctgc ggctcgactc gccattggct 145560 gggaagaggt ggcaatcagg gcgggccaag gcggctgttc tcgctccagc tcgatgctgc 145620 ctccccggcc cggttgcgct gtagccgctg ccgcctctgc ctgggtccct tcggccgtac 145680 ctctgcgtgg gggctgcctc cccggctccc ggtgcagaca ccatggtaag tgctctcagc 145740 cgggtgcggc ccgaacctca cccctcctcg gccggcctgg cagcgaggga actggccgcg 145800 cagccggagc ttggctcggg ggccctgggc gctcactgcc ggccacggga gcagcctcac 145860 tccttgccct cgcccagtca ggggaggtgg gaacgccgcg agtcgtggcg ggggcgatcg 145920 gtgcctttgc ttcccaggcg ggtttgccgc tccacaccgc agcttcctga gctcgggaag 145980 gggaggtgcg gcggaggcgt ggggtcttcc cggctctggc cggccccacc agtgtgggag 146040 gctgagcgcc agcggaaggg aaggctccgg gttcgcgtcc ccgcgctgcc cccgatgccc 146100 agcctctccc ggagcggtga caggtgagga gggtgggaag atagcgtggg ggtggggcgg 146160 atggtgacgg ggagaatcca ccagggattg gggggttgcg cccccacgat ccgactcctt 146220 tagtgcatag tacccctaag ggagaggagt caggggcggg gtgaaaggtt aagccagcgc 146280 catgggagca gaggcagcag cctcgccccc aagccgcttc tcaggtacag cgggtccctg 146340 acgctcaagt ctctccctgt ccccgcagta cggatttgtg aatcacgccc tggagttgct 146400 ggtgatccgc aattacggcc ccgaggtgtg ggaagacatc aagtaagtgg ccggctaccc 146460 tggctgtggc ccaggtcggc gcccagtgtg ggaggccccc cgcgcctcgc ctggtcctca 146520 gcctgctggc cgggtcgcgg gcgcgcatcc ttggaggtgc ctccgcgcct cgctcccggc 146580 tcgctgcagc tgcgctccca cgctccggga ctggaaccag gaggggaggg gcggctgggg 146640 cggggctgcg agggcgagaa ccgcggaaag gagcccctag gggtcaccac ggcttcccga 146700 cccggcctga gcgtgggaac gcgctgccgg cgctcccagg ctcagcaggc cgggcagctc 146760 ccacgcgtgc agagccccga gggaccacac gggcctccgc gaggttcctg cgcccggagg 146820 ccttagaaac aaacggaaga ctttggctcc tttatgattt acacacctgg cgtaagttag 146880 gcctttttgg tatgctgaaa cacgcgggag attatttctt gagcaacagt gaattaagcg 146940 catttattca tttttaaata aagtgaaaat tggaagtttt ttgttttccc caaatcccac 147000 tgcccacccc cccccgcccc aacccctatc ccccgttttt ttctactgtg ttgaagactt 147060 acagttctag gaggcattct actttcagtc ctggagcttt gagcccagta ttggggatgt 147120 gaacggaaat accacaccca cctgagagtt taaacctgtt ggaatgactg atgggtcagc 147180 atgtctgcag gcaggttgac tatttcactc taataaagtt gtctctcctt tttcctctca 147240 ctctcaaaaa atttgcattt aacttatggt acagctgacc accaccaact agctactcca 147300 aaaccctatt tatggatttt catattagag caacagtcag actatcacac tgtaacaaag 147360 gtggaaggaa gggtttcctg ggtgggcaga tgggaataca ctgttattat tcactctgat 147420 ttcctgtact tagtgatttc attttgttgt ctcaggtagg tgaggcttcc atgctgtttt 147480 tccctaagca cattacaatt attagcaatc aattctaaca ggccttatta tgcagttaca 147540 tgatggtgat ttctcataac gatctgtttc catagaaatg accagggtta tatattaagc 147600 acgaaaatgt aacttttctt tcccctggat ctgtatcact gagcgtttct ttcatgtttc 147660 agaaaccatt tgtattatcc acagggcata ttacctacat attataaaca gaagccgaga 147720 tttggcaagt atgtggttga atccaatgag aacataggca ccaaaagtta ctttaggatt 147780 tgaattttct ggccatctta ataccattat aatttttatt ttgcttagta gacagatatc 147840 aacaaggcag acatcaaaaa aagattgtta acagtataac ttaactaccg aattattttt 147900 aagccacttc ctttgtaaat ttataaatgc tatatatata tatattctcc atatgatgct 147960 tttgcatctg taagatagaa attcccaaat ttgctggtag atatgctatg acaatgaata 148020 tgcttagaat ttttagagca gaataaaaga tggctattat gattaaacag tcattatgta 148080 atttaaccag atttagattg tatatcctag ccattatcag tttagatcac tgttaaatac 148140 agagatgaaa aataatactt tatagagtaa tgtatcagac attttaaaaa gttaaaatta 148200 tccagagatt tgtttatatt gcttatattt tcccagagga agtccaaata tgtaaagatt 148260 caattgacga gaatttaact ctgcaaaatc aagctgagaa tattgttcca ttctacttta 148320 aaaaattatt tattaactta ctaatttatc tttaagtagc ttccaacagt attcataatt 148380 tgtctaaagg ataaaaattc caatcatcca caacacaatt tttctggtaa aaatgaaaca 148440 agttatattg gtcttttctt aaataaaatt gcctcaaggt tatgcccctg caagggaaaa 148500 actgttagtc acactggtgt tggtaatcca ggtgttagtg ttgattcttt atttgtctaa 148560 gcatcttttg gtggacccca gctgattttg ggtgtggttt agtgtctggg ctagagaaca 148620 tggttgaact ctctgcactg agggcattaa agctgctcct tggtttccaa cccaataccc 148680 ctgagtaatt actaaatgct ccctgggaga gcatttgctt tccacagaaa agaatcttgc 148740 tgcctgggga gaggggagga gaacctggag gagtgtaatt gaaaaatgct ccttgggtga 148800 ttctggttcc tcctcccctc cctaccctct gcttcaggga atattaaagc agtcattggt 148860 ccaagatgct ttcagtttta acttttataa gaaattgaat ataagaggaa aaacattttt 148920 ttttgtctca gtaagtacca aactcttatt ttttcttttg gtctttgaga aactatctaa 148980 attgatgttt atacaaattt acattatact tttttgcttc actaggaaat ggccatccat 149040 ccttcatgag aaaggatggg aaaagatgcc aaacatttaa tgacaaataa aaccctaagt 149100 aacagattcc agacagcaga cagaaaattc taagtggata agggccaaaa atgcttcatt 149160 gtaaactcag tatccactta cactcttacc aacaatataa ccatttcaca aaagctttga 149220 attcctctta agaagaagaa aaattgtcta gaccaagggc cagcaaactt tctcttaaag 149280 ggccagatag taaatgtttt aggtgttaca ggccaaggta aaactgagga tattatgtag 149340 gaatcaatat aactattaaa aatataacca cttaaaaata ttaaaaccat tgttagctct 149400 tggaccagga aaacgcaaac aaacaaccaa accagacaaa gtgggctaca tgtgatccat 149460 gggctatagt ttgatgacct ctgatttaga ccataacaaa tgtgaaaaag caaaagagac 149520 ctctgaatcc atttcaaagt tttttttgtt gatttcattt caatattcaa atagtccttt 149580 taagaggcta tgatttctaa gtaatgacaa aaaaattatc agggtagaaa ataaataaag 149640 ttaggagtga tacatcctag aaaagcacct aaatacttga tttcccatct ctgttatcct 149700 tccatcagcc ctgactaaac tcatcattgc gtcaaattac tgggtttaat tttagatttt 149760 ttctctgctt tctttccata ttttttctgg gtagcaacgg agggaagcag ttagagacag 149820 gtcttgctgt gtgcccaagc tggtctcaaa ctcctgtcat caagtgatcc tcttgactcg 149880 gcctcccaaa gtattggatt atatgcacca ccacgcccag cccaacttta gattttttaa 149940 cctcactgaa acatacaaat taggaagata gatccaaaac actgaaaaat cccaaatgat 150000 taaaagtctg taaagaaagt acactgtcct gacttaaata aatcagataa aacgtttatc 150060 tttaatgagt atagtcaagt gttttaggca ctgaggatag aaaagcaatg atgtaatcct 150120 taccttcaaa aaaaaaattc tagagatttc atgatatttt agtcttatta ttttgttttt 150180 attttcctgc ctataactca aatacggtaa ccttttgtct gttggatagc atatacttcc 150240 agttttgaaa ttaatgtgtc ttgttaaatt tgctaatcac aaattaatca ttatggttca 150300 aataaggaaa cactttgcta taaactagtt atgggacttt ccaagttgcc taaactttcc 150360 aatcctccat tgcatagagg aaaaaataat acttcacaga gctgatgtaa tgcattgaat 150420 aatgtctgca aaatgcttaa catagttctt gacaatagct taataaatga tgattttttt 150480 ttattttact ctagaatcag agtttttaaa ctttccgttc cccaatttac ttgaaagata 150540 ttaagccctc ctttaaaaaa tattattgca gtctatattc tatcagtcta aattttttta 150600 tcactctagg ataagtaatc atcatcatca tcatttttca tcattttgtt ttgaaatgca 150660 acaaatgctc ttaatatcca ttgtatcaaa gcaaattaaa acaaacaaaa gagcacatca 150720 ccatgttttg ccgtgtttat tcagattttc atatttttga aggtgtgatt taaacttcta 150780 tacctttctg tagatctgct taagtagtaa tgagcctatg tatgttttaa agtagtaagg 150840 atactttata ttataaagca atttcaaatt tccaaagttt tgttctctct ataattttgg 150900 ttgactgcta caacatccat ttgaaatagg aaaggacttt acagctcaat ttttttaata 150960 acaaagttga ttttccaaga ggttaattcc catatgtaat tacttattgt cccagagaat 151020 tacatatagg aaactgaaaa gtcagaactt cagcccttgt tcttctgttg ccaggtttaa 151080 tgcttctttt aagatataac tgtgaaatag gaactaatgt agtatacata caaaaatgaa 151140 accacattta ttttcttacc cagaaacttc ctcagtacaa ataaacacat taatatacag 151200 actaaggaaa caagtaaacc agatcattgt taggatcagg atccttttct gtagaaagat 151260 ctgccagtta gcctgaaata cagaggaagg cgttgcattg tattgtgtgt tggctgttaa 151320 gcttactaat gtttgaagtt atcttttgca gctggaactg gatgcccaag tgtatactcc 151380 atcacatacc tgctattgag ctctttgcat agggctcttc cagaacctgg accatgtctg 151440 tgagcagcat ggcaggcacc taggtgataa ggcgggtcat cacatgatct cagggagttt 151500 gtgggtgttt cttttgagtt ttattttggt tctgatgatc tgaggtatcc cccagaaatc 151560 aagctgggtc ttggagtctt tattttcctt gcattctgtg atttttgaga attaaaaaaa 151620 aaaatcagag gagtacatca cttaatttgg cgtgtcggaa ggatctttgc acattacggt 151680 gagcatcaga aatcagaaaa tctgtattct cactcttaat gtacccagat ctagttttgg 151740 caaacattta ctaagcattg ttatgatttg caagaacaaa gtcctttcta ctttcttgcc 151800 tttctccaga ataaggggct ctagcaaatg tttctcttta atgtgttgga aaactatagt 151860 tatgggttga tgctctgcag acatcaatag gaaaacacac ttaaggaaat gaatgctata 151920 aagaaatcta actggctaga tccgagggtg acagctcagc agacgtgagt acttgcagct 151980 ttttcattct ctcctttgcc tccttgttcg ttaaagagga aatggtggga gaaaaaatgt 152040 ctttattgtt tatgtctagc aaaatgaagc atctttttct gtgtcaaact atatcaaaag 152100 atagtgaagt taaagtggat tttattccag gtaaaagtag acattctgaa gtatttattg 152160 gacctcctcg ttactgtggc tttggttctg ttgcatattc tggtctactt ccccttcatt 152220 gttgaatcat gaaatatgaa attgttaaat ttattgaaat tctttgtggt caatcttcag 152280 ataacccaaa catggaaacc tttcactcaa cttgtcctac atacccttag cattccagcc 152340 ccatgggcct gtttttagtt tctcagccat accgagcctt aaatgtgtcc gtttaacacc 152400 tgtgcttgct atggtctcct tttcttcacc ctcccaaccc tcctgtaaat gtaaacacct 152460 ctcagctttg agatgtcaaa gcaaatgtca cttcttaagg gaaccttctc tcacatccaa 152520 acttttccct ttccttcata gtgctgtaat gcaagagttt gtacttagta tttgtactac 152580 tgattaatgt ctatcctcca tgacaatagc ctccataact ctttctagag tgctctctct 152640 ctccctctct ctatgtattc aataaaatat caggctctct ttctgtaata aaaatatgga 152700 tgtttatatg tgaatgaagt atctttcacc tttttcattt ccaaagaata gagcacacac 152760 atatttttat caaagattct tctctgcaac cagaatttat gtcagcatgg tgacatattc 152820 aactactatt taaatataag ctgttttcaa atagacattt ctttttctcc aagatttatt 152880 gagatataat tgacaaataa aagttatata tatttaaggt gcacaacttc atgttttgat 152940 gtacatatac attgtgaaat aatcactgta atcaagccaa ttgccatatt catcacctca 153000 cataggcacc ctcttttttt gtagtgagaa cacttaagac ctcccctctt aacaaatttc 153060 aagtattcat tatagtattg ttaactgtaa ttgttcattg taattgttaa tagtgacatt 153120 gctgtgccaa ataaacattt cttatatcag caaaggcaga agatagagca atatagcaat 153180 acataatagc aattcctttt ggaagtgtca gagttctttt atgttttctc atgtcatgtg 153240 gatgtattgg aaggactaag aactctattg ttagaatgac aatttcatat gtcaaaagac 153300 aattactaga ttggattcag gacatgattg aatcctttag caagtttcta aaaggcaggg 153360 atgtgtggtg ctgtttaaag tcagtgtcat agactgtaac tcagtgttta gaatactgaa 153420 attaaatttg aaatccagct gagctctcac catgtcatgc tatttcgaat gtaatatgtc 153480 acgtatatta aacacatacc atatggttct gcagtcacag tcctagatgc tttatactca 153540 ttatctcagt taatcctcat caccaccaca gaaaatagat tccttcatta tcctcatttt 153600 gcaaatgaga gaatagaggc gtaaaagaaa gcctgcctca aacactcagc tattaattat 153660 tagagctggg atttgaacac agataaagtt gtaattatca tgttatgtgt tttcctccaa 153720 agagaaagac ctataacctt cttctttcca aagacaacca atttagcaag gacatgtgcc 153780 atgtttatgt gtattagtac tgcctgctgc tttttctttt ttcttggaat gttcaagcat 153840 gcgtacaggt gatttttgac aacaatattt ttaaaggcga attgacatga agcatataca 153900 caagtgtcta agtaaatgca tttgtctgtt aaagctgctg aaaatggctc agcagctcaa 153960 atgaagattt gtaagttttg tggatatgtt ttcaaaattg gagaaattac cattaagggt 154020 ctcttgtgac tcaaaattgg agaaattacc attaagggtc tcttcctgac aattgcatgt 154080 caggaagaaa catgcaatta tgtatcaaga ctgttgttgt ttcccctaaa actaaggtat 154140 gggtgtcact caggagagca tttattgtta ctgtttacag tgattggatg agtgagtgaa 154200 cacattgaca tgaaaggact gacctttact aactaaaagg gggttcaggc aggaagtcgt 154260 tactggattt aaagggcctg tctctccact tggcagctcc ctcaagccag agtctctcct 154320 ttggggttca ggcttactga gaatcattag attaggctat tgaaaactta gaggctattc 154380 atgctccaca gccacttaga aaaaagcaat ataaagtttt acttttttag atgttatata 154440 tagttactat ccacagttta tagtgggaga ttttagtttt aataatggtt ggggacaatg 154500 caacattatg gtaccaattc agctcactac aatggaccat tctaggattt attatcaaaa 154560 cgacacctac gtactgtttt ttaatacagc atgtgttctg ataatgttgt ttatgggatc 154620 aatagtatct tggagctgtc attggtcagt ttgattgttt gatcctgatt gaccttttct 154680 gcccaagagc agaccttgtg ttgagattat gttttcaaca tgcctgtgtt caggctctcc 154740 catttcaaaa tactccatgg aaaacttctt taactgaaat atagcagtct tgtcatattc 154800 ttaacttact ctaaaaatgt acagtagcag gagctcaggg tcagaatatg aattcagttt 154860 ttctgtgttc acatattgta gttaaggaat atctccttgt atgtgaagtt ataagaaatg 154920 ttacctggat attttggaca aattgtatac ctagttttaa aaaataaata ataaaaaata 154980 ttctttagtt tgaaagcaaa tgatggactt ttgttagtaa tgtttgatta cttgtttgtt 155040 tgtttttttt tttttgtagg gagcttggaa atgtgtgggg aaattgattt cattaattca 155100 ttcactaatt tttaaacatg tataccttta ttaagcacat gtgcaccttt attaagcaca 155160 tgtgcaattc caggcacctg ggttaggcac cagtaattac attaaagaaa aaaatcccaa 155220 cttaccaaac accaccacta taaaaatacc aactacctct ttgtgaaaag gtaattagta 155280 taaataaagg atctgcttaa attccaacaa ataagagtaa gtaggaagtt gattgctact 155340 gggaaactat ttcacttata cgtattcaac ttctaaacag ttactgtggc tacagataca 155400 aagaatgtga ttatttctga ttttgttgcc aggtgagaat tcgatcttta tttttaaagc 155460 ccttaggggc gacagagctt gtttgtttct tggctatttt agtttgtaaa ttctatcagc 155520 aaatctgcat actaagtgca ccttaattat ttgattctga ctaaaaatga ctaagatttt 155580 gttgtcattc ttcccttctc ttcttccact tgccatttcc tctttctcct ttttcccttc 155640 ttccttcatc ttctccaact actataactt accagttaga ggaagtatca ggcattaagt 155700 gtgcaagaag tactaccctg agatttttgc ataggttaat tctgctaacc aacacaatga 155760 ccggaggtaa ctgctgctgt tagcctgttt tacagatgag gaaatgaaac accaagagtt 155820 gaatctgccc aaactgcaca tgggattggc ttagttgttt attttccata ctgtgtgagg 155880 aaagactttg acttatgaca tagatgaaat caagggagtt tctctttatc taggtttact 155940 tgttagccaa tgaaagtgct tttaaacttg aacctctcta aatatttatt attgtctgct 156000 gacctctttc attgagtaat tcactgaata gaaaaattta acgcttaagc caagcagagt 156060 cattgggtat gtaaatatgg caagtcatag tatttgaaaa tagtgaaaga ctatgttcac 156120 ttgggatttg gtaggacttt taaaaataaa aataatagta gtaagcagca acagactgta 156180 atacaaacta ggatgcatga agattgtagt gctgttggtt attatttgat agatgagcac 156240 tcttgaacac agtttaatcc aatttgctta gagtttatat gatatttttt gtcatctaag 156300 aagcattgaa aaaaatgaca gtatgtccaa aattttcaga aaaatcttaa attgtggttc 156360 tcagggaaac ctgtggtttt tctcacatgt aaaataagta caatttaaat aataaggtaa 156420 ttgcataatt tccctattat gtaattatat aattacataa taataatgta acaatgtata 156480 cataatgcaa caatttaaac aaaattggag ctcaaaaaat aattctgaaa gaatgaatga 156540 gtctataaaa tttggtgaat gtacttggct gaatttggat aatttaagcc catataaatt 156600 ctacttgtca caaaaataaa ttttaaatta ctagaagtgt tattcttgcc tttgctcttg 156660 aggaaagaac ttaaaagtta tgggatagca aagtgggaga tgcagaatta tgagataata 156720 ttacttgaat tatgtaaatt gtttatgtaa tctgaatatt agcttacttc ctaacttttc 156780 ttaaatttac tggtcgccct ctgtgacttc aggtagcaga taaaaaaaca gccctaaagc 156840 taaggagaga aggaagcctg cagtgtggga agggaagcca ttaaaacttt cctcaaagaa 156900 cagtggcagc tatattttgc agagtgctgg gaaatggtta ttatcacatc tctgtgcttt 156960 ctggaggtta aaacatttga cagcatgaag tccgattata tataaattta cctaaaagtt 157020 agaagaaaag ttgtattgag aattgagaga caacagagaa ggcttggttg attcaaaatt 157080 tctactagct tcaaaggcca tattatctga aattaattct cttagttatt attccttcat 157140 tgtatattct ccatctcagt ttagtgtatt aaactgcttc atactaaaga gtttgcgata 157200 cctccaatta acttttgcag tatttccaat tagcttttcc cctgaaagga aagaagatga 157260 agaataggct agagaaggaa aggaagtggc cccagaaagg ttaggtataa ggtggtaagg 157320 atggaggttt cctgctcaag tccttgctcc ggcgtcactg tccccatgaa gcctaccatg 157380 aacacctatg tatattatcc tcatatgtat actgcatcca gccccttcct cttgcatttc 157440 cactctcatg tctttatcca gagctacctc ttcctgtagt atctattgct tcataacata 157500 ttatatcatt ttgttatata tcatgtttat tgcttattac ttgtatctgt cagctaaaat 157560 ataagcttta tgaggacagg gatctttacc tcttttgttc attgatatat taaaagtgct 157620 tagaaaagtg cttggacata ataggcatcc aaatatttgt ggtaaaactg ttccataaga 157680 aaaaaagaaa aagaaaaagg gtagccccac attcccaaag atgtaaatag agtcctaagt 157740 actttgaata attcaaagga attcagagtg aaatttttaa aagaagatta ttatttcctt 157800 ccatgtacta ctgtttaatt tctatttcag tccccactgg ggagcattga cactggtcat 157860 ttcaggaagt aaagctttga tgataaggga gcttatttta ataaagagag agaaaggaaa 157920 ggaccaatgc attgacccat ggttaggggg tttctgagga aatggctgtt cccctgtttt 157980 atgcagaggg actgtggatt ctgcttgtac cttgtttatt tatgggttac tagagattgg 158040 tgagtgtttc aaataaacag agtttattac agaaatttat gtgatttgat ttattccaat 158100 tggaagaaac agaatggtga aacatgaata aatttctact tattgtatct ttaattagaa 158160 tattctgtca gagtctccaa aattttaatc tatagatcag tgttgtttca aaataaaatg 158220 ttaatagcaa aatgacaaaa cataaggtat ccatatccgt caaactattt atcccattcc 158280 taggaatttg ttctgttttt acatagaagt gtaagaagct ataaagatat tcataacatt 158340 ttttatactt gtggaaaaaa taaaaacatc taactaccta ccaagacttc aatatgtcta 158400 caatgcaatt ttatgcagcc atggtataga agtataatta tgatatgaaa agatatccat 158460 caaatattca ctcaaatggg ttttaaagca gtatgttcta tatataagta tattcaagca 158520 tgaacaactt gaaaattata atagcagtga ttatctcagg atagtggagg cttattttct 158580 ctttttagtt tttcctttcc tgtactttat atatattttt acaattagaa attagaatgc 158640 agaaaatata tagtagcatt tatcagagga taagacaatg tttgtatatt acgttttcat 158700 aattttttga tatctcactg acttttggta ttaaaaagac ttgaggtctt cgaagagttg 158760 tgttgctttt gtttaacagc tgtttatgtt tggaaaaagg gagaagaatt taatatttta 158820 tgtacagtgc tataattgtt tgagtagaca tggggagtgc tatattcact gggaatatgt 158880 atagtacttt aactgcttag aatatacttt tgtacacttt atctctttac tgttgtgaaa 158940 ggtgaattat ctgttgcttc cacttcagaa aagagaggtt ttgtttgttt gtttgttcgc 159000 ttgtttgtta gctgagtcag gatctggctc tgttgcccag gctgaaatgc agtggcaaga 159060 tcacgcctca ctgaggcctc aactcccgag gctcaagcaa tattctccca cctcagcctc 159120 ccaagtagct gggactacac acttgcgcca ccacacctgc ctaatttttt ttttatttct 159180 aggagagaag aggtcttggt atgtcgccca ggctgttctc aaattcctga gctcaacgga 159240 tcctcccacc tgggcttccc aaagtgctgg gataacaggc atgagccaac ctgcccagcc 159300 agaaaagaga gattttaatg cttatgtgct ttatgtgatt tgctccggtt catttagctg 159360 gtaaagcctg ggctctgaaa tgcagatctc tgtgcttaca gaaaacatgc ttttaaaaaa 159420 aaagttatga ataaatgcag gcaaataaat tataagctct gaaggacaga ggatgttttt 159480 aagactatct gcatactaag gtatggatgt aagacatacc accatttgga atttttccct 159540 tgagcataca cagatatagt catgagtggc agctgtgata gcacagcttc tgaaggagag 159600 aaagcaacta aggactgcct tgaaaatatt taaggagtta tttgagctgc tgtgttgcaa 159660 gttcgcctaa ttagagtggc tactttcctt tgtattctct tggcaatgta cagggattcc 159720 gatgaattgg attgagagta agaggttgac atccaagctc catgtatttt tgtgaagtta 159780 acattggcaa tgatatctgg aacttagttc ttttctacag ttggccatgt gggttttttt 159840 atttacatac atttatgagg tacatgtgca atttgttaca tgcgtagtag gtggcagtgt 159900 tttaaacaat ggatttcacc tctcctttga atgcttgttt gagaaaagta attcaaatcc 159960 tatctaaata aatacattta tataatcatg acaactttta aaaacaatta taattttctt 160020 tcactagtac tgtgtatgtt ttgagctact gtgtttaata agctaaggat atatttgaag 160080 attttatcag tccacttggc atctcaaact taatatgttt aaaaacatct caaacttaat 160140 atgttgaaaa catagctctt gaccttgacc ccttgtccgc ctccaacaag tctttcccaa 160200 ttcagctact ggtgacaaca tccttttagt tgcttagacc aaaaacctca gtgtcctcct 160260 tgagtcctct cttttcacac cctgtgtcca atccatagca aatcatgctg aatttacctt 160320 tagaatatat atgagtttga ggcatttctt acctgctcca cgggttcacg ccattctcct 160380 gcctcagcct cccgagtagc tgggactaca ggcacccgcc accacgccag gctaattttt 160440 tgtattttta gcagagagac ggtttcaccg tgttagccag gatggtcttg atctcctgac 160500 ctcgtgatct gtcagtcccc gcctcccaaa gtgctgggat tacaggcatg agccaccgcg 160560 cccagcccac caacacttct tacctgaatg ttggcagcag cctcctaact ggtctccctc 160620 ctctgtcttg taccccttta gtctattttc aatgcagtag ccaggtgaat ccctttaacc 160680 tagagcagtt tatatcacca ctcatgtcac aagtgctcaa gcccttcacc tctttcaagt 160740 aaatggcagt tatcccaacg tccaaccagg ccaagcactg tagcctcatg ttacctctcc 160800 tacccgttcc ctgatagcta cttttccccc tcctctgttc cagccacatt ggcctctgct 160860 gtcctctgac accccaggca tgctcctgcc ttgccctttg cacttgctct tccctctgcc 160920 cagaacacaa tctctctaaa ttgctcgtgg cttactctcg gagctccttc agactttctt 160980 caaatgccag cttctcagtg aggccttcct ggctgttgtc cttgagctct ccactgctct 161040 actctgcttt atttttctcc aaagtataat acttaccatt atatgttatt tattaatttt 161100 atgttgtgtc tgttttccca gaatatatgt attgcaagaa gtcagggatt tttgtctgtt 161160 ttactaaaga ttgtacccct agtacccaga acaatgtctg gcacctaata gggactcaac 161220 aaaacttgct aaacatctat taacccaaaa gttatttaat ttattatatt taatggcagg 161280 tattgtattg agcagtagag tactatgtac tgttaatctt gctaaaatat acttaaatat 161340 atcattttaa tttagggagg gggatatgac atcatttctg ctctctcact agaattttag 161400 gtacatgatg atgtttaaac aaatttgccc aaaaaaacca aattctattt cagagataaa 161460 gccattgttt atacaattat tatacttata gtagctattt ttttaacttc aacttttctc 161520 ttctgtcttt cttgtttttg ttttccagaa aagaggcaca gttagatgaa gaaggacagt 161580 ttcttgtcag aataatatat gatgactcca aaacttatga tttggttgct gctgcaagca 161640 aagtcctcag taagttgaat gcaactttcc ttctttggcc aagttacacg tagaagctca 161700 cagaatgcat ggttcaagat cacaacgcag ggttacagaa gtggtgcaga gcatttgtac 161760 aacctgcata gttgtgtggt gggcatccac atatcatgtt aggctcaggc tatgccaagt 161820 cttatttttc cttttgcaaa ttctttagag aatagaaaag agtaaatgtg ctcttcttgt 161880 tgttttttga gacagggtct ggctctgtca cccaggctgc actgcagtag tggcccaact 161940 ccggctcact gcatcctctg cctgccaggc tcaagtgatc ctcccatctc agcctcccaa 162000 gtaactggga ctacaggcac gcaccaccac gcccagctaa ttttttgtat ctttggtaga 162060 gacgaggttt catcatgttg tccaggttgg tcttgaactc ctgagctcaa gtgatctacc 162120 tggctcagcc tccgaaagtg ctgtgattgc aggtgtgagc ctccacgcca ggcccagtcc 162180 tttgtctccc ccaacagaaa gccatgtatt caggaaggaa agagaattat tgatacttaa 162240 tgtaacaaca tcagttgtgg tgtcaagaaa acggtagtta ttcaagtata atatgtgttt 162300 ctccaaatct cttttgtagt cacaagttat attaactatt ggtaaaagaa acatgaaact 162360 gagatctacc acagataaat atctttctga agaaggcaaa gttttaaaaa tttcatggag 162420 attctcaaag tagtatctat tattcccaca gtgcttaatt tttaactggg ctatagtttg 162480 gatacaatag aatgaagctt ttagctttgc cacatataac taggaaagca aacactatct 162540 atagtagaat aaaaattgtt ttaaaagatt ggtaaatgtt taatattaac aagaaaaata 162600 tctagcctcc agcaatcaaa attgaaacaa acatgaaata acttttttta ttatcaattt 162660 ggcagtctta aaaacggaaa tatctagtgt aatgatggag tacagttaat gggaaatcac 162720 atacatttta aatgtgatgg tagttaagac aacttttttg agaacaattt tgcctcgtga 162780 aagcctttta aaaaaatatt aaaaccttca gtaatttttc ctaaataaat tattagatct 162840 ctttccatta agatttatgt atgtgttttc atgtcagagt tccatatatt atcaaaaact 162900 gaaaaaaagc ctacattttc aataagaagg gattggttaa ataaattatt gcatccatat 162960 gttgaataag atagcaatta aaactgatac acagcacttt gggaggccga ggtgggtgga 163020 tcacttgagg ccaagagttc gagaccatcc tggccaacat ggtgaaaccc cctctctact 163080 aaaaatacaa aaattatcca ggtgtggtgg tgggttcctg tggtcccagc tactcaggag 163140 gctgaggcag gagaatggcc tgaacccggg aggtggaggt tgcagcgagc cgagatccta 163200 ccagtgcact acagcctggg tgacaaagtg agactctgtc tcaaaaaact aaaaaaaatg 163260 tgatacagaa aatttaagtt ttaggaaata ttaatgattg tttattaaga aaaaagcagg 163320 acaccagggt acatgttata tgtaatcaag ttattaaata tacatattta tataaagact 163380 tttgtatcaa aatattgtca tgtgcattct tgatgatttt aaaaatctgt gttttttgga 163440 aaaaagaagt agtatatcaa atatatgctt taaaatggtc aaaattaagt acagggtgag 163500 tatctcttgt ctaaaactct tgagaccaga agtgtttagg ctttcagatt ttttttggat 163560 tttggaatac ttgcatatcc ataagaagat atcttgagga tgggattcaa gtcgaaacat 163620 aaaattcact tatgtttcat atacacctta tagacatagg ctgaaggcaa ttttgcacaa 163680 tattttaaat gattttctgc atgaaacaac attttgactg caatccatta cacgagttca 163740 ggtgtagaat tttccacttg tagtgtcacg ttggtgacca aaaagtttca ggttcttgag 163800 tatttagaat ttcagatttt cagattaggt atgctccact tataatttgt aatagaagct 163860 gaaaaactgg ttgctttgct tgtaaatgca aatgtacacc attaacatgt ttatatgttg 163920 tcttctctga tttcattgta ttgtcctggc gggatttttt tcatatgatc tatgcttgcc 163980 atgcacaaaa attgaagaca ttttacatta acttaataaa cacttatctt cattttttct 164040 attaacaata tttcacctat tatatgcttt ttttcccctc ttgaatttgt aaaatagatc 164100 tcaatgctgg agaaatcctc caaatgtttg ggaagatgtt tttcgtcttt tgccaagaat 164160 ctggttatga tacaatcttg cgtgtcctgg gctctaatgt cagagaattt ctacaggtaa 164220 gcaatttgag gtcctatagt taaaagttct gtgtttataa ctctcaaata tagactctag 164280 aatacttttg ttcactcagc tgtacaagag tgtcttgctt ctgaaatgga atcgtagtca 164340 cctttttcta attcacataa aatcatcgtt attggagcgt ctcgaataca ttttagcatt 164400 atattttttc tttaacttct ggttgctgat actgaatatg caagttgtaa attaatgttc 164460 cctgaaaaac tccaggtggt cttttgtgat ctgttcaact gccctttttt tggggtaatg 164520 gctctatatc ctagtgattg tggttttatg tgttagacat ttaatatggc cttattttct 164580 atttacaaaa cattcctagt ttgtagatat aatcgagcaa tgtatcagta tttatgaagt 164640 tctacctgga gctcaatgtt taggaaaaag tagtagggtt ttcaggtgtt ctgtcaccaa 164700 aacatactta agatccatta tcattagtta aatattaatt aatctactgt acgagggaaa 164760 ataggaaaca gtccaaagtc agtttcaggt gaaaaagtta aataatgcaa tcacaggcct 164820 ttgtcttaca ttcttgttaa attaccctta aaagattgta ctagtaaatt acacattaga 164880 tgagagacta tttctggatt gactatacta ttttaagcat tgacatttgt gagaccctct 164940 tcttgaaatg tgttaacatt gaaaattata tagttgtttg caaaggatat ttctaaactg 165000 gctgctttca gcagccagat cttgctggaa aatgatgtca gtgcaatact agctgaaccg 165060 aagcccagtt ttgagggaat gtaaactact ttcaatccag ctgcattatt gatttaacag 165120 ccaaaataag aactttagtg tttaatcaaa atgtctgttg tttacatagt gacatttttc 165180 ttataaactt aaaacagcat taaatcaagt tggaaactgg tttgcaatct cctgccatgg 165240 atttactaaa ctactggtgt caaaaaaagc tgccacttcc aaatgaagta gctgccaaga 165300 gatatttaaa taaaattgaa caggcagcat ttaatgggat tattttaaat ggattaaaat 165360 ttgatactta aatgttttgg gggattttgg gtttttgaat acatatttca ctttttagta 165420 cttttgttgg ccactatcaa cttatagcaa agtttcaact ttcagttgtt ctgacttaca 165480 actctctctc tcaatgccca tgtttacata acaaaacaaa gattcaagtg gttttattaa 165540 ttcctttttt ttttttttaa cctctactca ttgtgaccat ctgctaagct tgttatggca 165600 cagtaagaga gcaagcggcc ttttaggtgt gtttctctgt attccagagc tgctttatcc 165660 aataggacag ccactagtgc atgaggctac tcagcattta aaatgagatg tgcaaactgg 165720 ggagtggctc acacctgtag tcccagctac ttggaggctg agatgggagg atcacttgag 165780 cctgggagat ggaggctgca gtgagtgagc tgtgatcaca tcactgcact ccagcctggg 165840 tgacagagtg agaccctgtc tctacataca tacatacatg tgtacataca tacatacaca 165900 caacaaagtg caatgtgctg caagtgtaaa atatacacca gatgtcaaag acagtacaaa 165960 aagaataaaa gatggctcat tcacaatttt atattgatgg atttcatgtt aaaatgttaa 166020 tattttggat atatttggtt atataaaatg tagttaacat agctttaaat tatacacaat 166080 tttattttat ttctctttca acgtgagagt gaaaaatgaa gaaactagaa agttagaata 166140 ctgtagtttt catcaatatt ttacattatg attcagttct tttattttct cctggttttg 166200 aaattataca ttttaagggc aaattgaata tgttaaaata ctgcttttct aaaaagtaga 166260 tatttatgtt atcattaggc tgattacaca gaaattaaat ggactttttc tgtgtgacta 166320 taaaacaaaa tcattaactt tcatacattg ttttaacatg ccagcacaca tatattgaca 166380 tttttattac gtaaattagt aaaatattct ttatggtgac ttcaactctt cactaaaaat 166440 ttaaagactt tctttctgtt tcagtcctgg ttttaatcag tcacagcatc tttagattct 166500 atttcccaaa tactgagctt tgggaattct gccacttttc tctatttccc cccacacttc 166560 tttgggagag tctaccatta tctagaggtt gaatggattc cctgcttctc ttcatgactc 166620 ctccaatctc ttctccatcc tataactcta gtgctttttc tgaaatgcaa atgtctctcg 166680 tcgtatcgtt ttcttgcttc aactctggag taatttttta ttgctcactg gaaactgtca 166740 aaatatttaa aacaattgaa acctacatcc tttctgcatc tttcctctcc ctctccccca 166800 gtccagccaa acttaacctc tttaagaacc ttgtatttat cttgttcccg ctatcctctc 166860 agcttttcca ctgctcttct ctttgcctgg agcatgtttc cagcactttc cccatagata 166920 aattctatta attctgcagg taaaattact tctctgggaa gacgccactc ctctctccaa 166980 gctctgccct atcctagtca cggtaactaa cctctctctt attttcatag gacactctat 167040 ttccccagcc acagtagttt tcatactgca ttatcattcc catttgatgg actggaatgt 167100 aaacttcagg aagccagaga tcatgtataa tctcttaatg tttgtacctt cacagatgca 167160 atctgtatat ctgagaataa atgaatatct tttttaaaaa agtgaatatt tgttgtttgt 167220 gcaaaagcag aaaacacaca ataggaagca aacacaccaa tagatcaagc tgaattattt 167280 gcctcaggat aacccatttc gagtgagatc ctgtaatttt tttttacgga tgaatcatag 167340 attgaaatat aaaagaagag acatacacta gaccaatttc cttttcaggt gaacatccta 167400 aattactgtt tattatttaa tttacagggt acattgcttg tatctgttga cacttttaaa 167460 atggtcgttc tgacatttcc attttaagct tttagggaac actaaaatag ctactgaaat 167520 cataacatgg gagactattt gattatgcct tagctgagca actctgactc actcatcata 167580 gcaagcagaa tttccttgga agttgcctgt ccacagcctc tcagttagat acattattaa 167640 gaataggtct gtcttatata tgtatataca taccatacca ttgtttttac ttttcttgaa 167700 ataatgaaag aaaatttcta agtactagac acagcatata ataggcaatt aatgaaacag 167760 tctttaaata atagaattaa ttaattctga agtatatgtg tctttataat tatttggtcc 167820 agcctgtttt atcaggataa tacctctttt gcttaaattt tttattcgag gatactgtat 167880 agataatatc tgtagcttag ctccagaaaa atatttgttt tctcacatgg aggttatcat 167940 ttccattcac agttgactta acgaatacta acatatcttt ccccttgaaa ggctttggtg 168000 ccaatatcca attcatgacc ctgcttatgc agtctctcct gtgcttacaa attcaagttt 168060 aaaaaatttg cattatgcgt attttaagcc acactgagac tttaacaaaa aaatagctag 168120 aagtcctaaa tggataaata aaatataatg ggatatattt aggataaata aaatttagga 168180 taaataatat ttaggatata tttacgataa ataaaattta tcctaaatgg ataaataaaa 168240 tataaaattt aatggataat aaaatataag aaatattttt agagaaaact ttgtagaaat 168300 actcctcttc tctaggcatt tcacagttta gaattgattt tttcttaaat caccagtgaa 168360 agcatcaact tgtgggctga tgaaaaaagt aataatcttt ggcatttatt ctttggttga 168420 tttaataatt gaaaatataa tgtatcaaaa agagaaaaag aagacatttt taaaatttga 168480 tttttggatc ttatatattt tacctgggta tattgtttta aagacaagac catatttcag 168540 taagaatttt gtctagtttt tccacttttt tattcataaa ttgacaataa aatgtgtggt 168600 tccatgctat tgctatagtc aatgtctttt tttgcgtttt ttaatgtttt attattatta 168660 ttattattat tgagacggag tcttgctctg tcgcccaggc tggagtgcag tggcgcagtc 168720 tcggctcact gcaaactccg cctcccgggt tcacgccatt ctactgcctc agccgcccga 168780 gtagctggga ctacaggcgc ccgccaccac acccagctga tttctttttc ttttattttg 168840 tattttttag tagagagggg gtttcaccgt gttagccaga atggtctcga tctcctgacc 168900 tcgtgatccg cccgcctcgg cctcccaaag tgctgggatt acaggcatgg gccagagcgc 168960 ctggctgcta tagtcaattt ctatctgaag cttctagtgt actgatgaaa agactgcaaa 169020 gaatacatgg gatttgaaag cttcataaga aaatagctga taaggaatgt tcagtttaat 169080 gttatttcta ccttaatcca cgctcacctc tactctctaa tgtcctattg cattatggca 169140 cattctattt tccctaaatt actgcaacag tctctcccat ctcacatgca tttctgtagt 169200 atgattttgc ctcttccttg cgggaggtaa aattctcttt tccttgaatc tgggttggct 169260 ttagtgacta tttaaccaat cgaatggggc agaagtgaga tatttaaact tcctaggcta 169320 gagtacagga agtcttggtt tccttcaggg tatcttggaa cagttgctgt tgcgatggta 169380 cctctagaaa cccagacaca tgaaatgaga agcacaagcc acatggagag gccacgtgta 169440 gactggcaga tagcagttct agctgggctc tcagcaaaga gctggaaata actaccagcc 169500 atttgactga tccatcttgg aaaaccagcc tgctccggca ttctgataat ttcagcccag 169560 tttccatctg attccgtccc aagggtagaa ctttctaatt tagccagaca acccacagaa 169620 ccatgaaaaa taacaaataa attattaaag ttgtttgaca tacagctatg gataactaga 169680 ccactactct gtgcattatt tttcccttta tattttatgt ctgtgttaat atgtgtgtat 169740 atgcttgtac aatgagtcaa gagctttgag aaagctgtat ttgataagat aatctattac 169800 ccctgggaat aggagccatg tcagtcccaa acagggctac caattcaatt tcgatggtat 169860 tacattatta ttacattata ttgtttttac ctcccctaca aatttacaga gttaaaatgc 169920 aattaaaatg agcagcgaag gctgccaggt tgactatgaa aatatacgtg ggcaaatgca 169980 attttagaga acagttagga tttaaattat gacctaattt aaattagaac tcaggtgaat 170040 agttagatca gcgttctttc attatttgct ggagacactg gcttcttcaa ctaaccttga 170100 tatgttgaca gtttcagtca ttcactcaac tcagatatat ttgagtttat caactagcgc 170160 ctctgttcag cctgccatgg gattttcttc aattcctgtt ggctccaggt tttgacttat 170220 taaatacaac attgtgattt ggccaccagg aacaatctga gattgatgac tgatctaatt 170280 cttaaatgtt tgtgcaaagg gttattttag gaaatatgag gattttcctg aacagctgta 170340 aaattctaat acttccctaa attatttata tttcttaaga aaaaagagca ccattcactt 170400 tatttttaac tttggaattt taaaaaagag ctgttttctc agcatccaca acgccaattt 170460 ctataatcca ataataaaaa tagttttatt ttttaaaaac ttatgaagga agataagaag 170520 tattccagaa ttgtaaggct tttcagtttg aataactggt atatattttc ataatttttc 170580 aaaatattga ttaattgtta catgtagtat tatatagact ggatcttttt taacatgagt 170640 ttaaaagcaa tagaataaga gaaatatttt aactgttttt ctttaattaa aatatttcct 170700 tggagaggat ggtaatgata aattgatata gctttaaagc tatttccatt taaataattt 170760 tagtgactct ggtacagttt ctttttaaat agagattttc aatgtttctt ttagaaaaga 170820 atctctatat cttttcagtt gaaaagctat ttaattctca agaagcagct ccaggaagaa 170880 aaggcaggaa aagtattatg aaagcattca cttcctactc caatctcatg ctatttactt 170940 tggtgtttct catcatctag ggtttgatgt tccattttac ttaggtcgac tttgtttaaa 171000 tgttttatct tcaaattcca aaatgacttt tttcttataa aacttaacaa aatatttttt 171060 aaattacatg actctcatta aagaatgttg gtgttggtcc tatttttcag aggtgtgagg 171120 gatagaaaga aaggaaatac tatacctcat cctccttcat tgaaaaacaa aaagaggttt 171180 gcaatgactg gttttaaaga tttcttgcag atctaaactg cagtaatatt ctcaacacag 171240 acaactccct tctccccatc cactaaaaca aggattcttg gatactgaaa atgtttttct 171300 ttaaaacgtt caaggcaagg tcttacccat ttctgtggat cctttaaaaa atttaattag 171360 atgtccttga aacaggaaaa tcaggtagtt gacttctggg gactttgccc ctgtaattat 171420 gtaagaactt atttttggca tgcatattac aaaaagaaaa tgtgcattta ttttgaaaca 171480 tcccaagatc tttaaaaatg tgacacttta ttactcaaag gaatgataaa ttgtcttttg 171540 ttttctaaat ctgtgaagtg tactatgtct gaaattccag tagagtaccc cagaatacca 171600 gaactaattt ttcttttata aatgataaat gcaaggtgat aaaataaact agactgacag 171660 ggagcagaat tcctgagtag aaaaaccaca ttcatttgct tgtagaaaaa caataaaatg 171720 atattagaac attaagaaat gaaattatta aattaaaacc tttaggtatc tcaagatatc 171780 aaaatagatg aatggaccta ctctctatat ttttcctgtt ttttgatttt atgaagccta 171840 gaagaatgaa tcaaagcata tttcagattt acatcattgt ttcctgggaa tctacttgtg 171900 ctgcaatata agagtttaca ttttttcagt gtctttttta ctttcataca tatggacata 171960 cttctttgca attggccaca cagctcttta ctttctgtaa gtgcttctgt gggtatattt 172020 agaaataaca cagaatatca gttttgatat gaaactctta ttccccagaa tacattgtta 172080 gagcaatgtg gatgaactat catctttgtt tagatagcat tatcaaatgt agaaagggtt 172140 ttctcttggc cattgtaaga atctttttgt agcaaaaaaa aaaaattgta gcctgtatat 172200 gaggatgaca catttattag caatataagt gtacttaaat gaaaataact tttaatcatt 172260 aatctcattt cttgtaatag tctggctgtg gtaattactt taaattttga aagagtaaag 172320 ccaagtaagg aaggttgtaa ctcaagaaat cccaagagta ttttttatat tagatactat 172380 ctaaagaaaa tcaatattac attaagtggt gttcaatctt ttatgattca ggagagcact 172440 ttacttgttc aattaactaa tagtaaattc aataacacag taaatctctg gagagttggt 172500 tactgcaaaa agacagaaaa ggtgaacatt ctcatttggt gtttttggta tcctttggtg 172560 agggtgcagt gcctatcttt agtgactcag gaagtactga cttggatgat tgtgtccact 172620 ctctgacttg ccccagcata agtggtgcca gtgacataag tggtgcaagt cgttattgcc 172680 ttatgccatc tgcttgagtg aaagtagaaa tgttttaata atccagtaaa aaagatgagt 172740 atgaaaatga tataagcaca ttggtacatt gatttatgaa atatgtaagc attctgtata 172800 gaaaaaatat taaaatgcct ttttgaagtt attgtttggt tcaatttcct tttttttttt 172860 tttttttttt gaggcagaat ctcactgtgt cgcccaggct ggagtgcagt ggtgagatct 172920 gggctcactg caacctccac ctcctgggtt caagcgattc tcatgcctca gcctcccaag 172980 tagctgggac tacaggcatg ccccaccacg cctggctaat ttttggattt tctttctttc 173040 tttttttttt tttttttttt ttttgagatg gagtctcact ctgtcgccca ggctggagtg 173100 cagtggcgag atttcggctc actgcaagct ccacctcccg ggttcatgcc attctcctgc 173160 ctcagcctcc tgagtggctg ggattacagg cacccaccgc cacaccctgc taatttcttt 173220 ttttttctct tagtagagac ggggtttcac cgtgttagcc aggatggtct tgatctcctg 173280 accttgtgat ccgcccgcct tggcctccca aagtgctggg attacaggtg tgaggcaccg 173340 cgcctggcca atttttgtat tttcagtaga tgtggggttt caccatgttg gccaggctgg 173400 ttctcgaact cctgacctca agtaatctgc ctgcctccgc ctcccaaagt gctgggatta 173460 caggtgtgag agcacgcctg gcctcaattt cttttttaag tgagccaaca acacagtacc 173520 tgggcctgac tctattagaa acattttcta aatgcagcca caagctcaat tttgactttg 173580 tgagaaatgc agataaaaat aaaactctct cagtgatttt ttttttctca cccgattttc 173640 tttcacaaaa tgtttcacaa cataagtaga agtataaggg gtctgagagg gctatgaagc 173700 agttctcagg tcgtcccttc taagggaaga gtcctaatga gaatgattca tcattggaag 173760 aacctctcat actattcaac caagtaaaac agttttagga ctccggggtt tgtaggttat 173820 atatacctct tcaatactta gtggctgcca tattggatta cttttacatg gcaatagcag 173880 cctctagagg caatccggat gaactgatgt ttttgctaag aattagcttg actccatcag 173940 gtataattgt ggggaaggaa acagtagatg cagacggttc aaaaagtcac cagatttatc 174000 gagttgagga ggtcattgaa tcatttatct ggattagatt gtcaaacttc atgacctagc 174060 tcaaatcaca tcttcagttt gaagtatttt taactccatt ctttcagtct gaattgttgg 174120 cttcctcatt tgctcatcaa aagccactct acacatccat ttgtgatata aaccatgagc 174180 tgcttaatcc tcatcatgca gatctgcatc caaacatcta ccctgcaccc aagcccacat 174240 gacacagtgt atgatatata atgcacatac aataacatta tagagtgaat gcatgattga 174300 gtggatgtgt ggaagggaca gaaaattaat gctgtaagtg tcctgttaga gtattgctca 174360 aatgtggaca cccgcggaat catctcattc tcaccgtagt ttcctcccag taatctcact 174420 ggatttttct gtttttctaa attgacctct ttgagacatt tggaatgaaa gccatagttt 174480 aattttggca tatctccagt actttcattt taggtatgtt cttttatgcc catgacattt 174540 tgaaactgtt ttgttgtttt tcttttcttc tctttaaaaa taatcaccag ttgtgaatag 174600 attactactt gcttggtgta tttgtttttt attgctacca taacaaatga ccacaaactt 174660 aagtgcttaa cacaaattta ttaccctatg gtttcataag ttggaagtcc attgcaggtc 174720 ttactggact atattcagcg tgctgtgctg gtgggacggg actgtgtttc tttatgttgg 174780 cttttgggga taattgtttt cttcctcatt caagtattga cagaattcag ttccatgcag 174840 ttatagggct gaggacccag tttccttgac tgtcagcagg cagctgccct aaagtcctgg 174900 agacctctct ccagtccttc aacagtctcc tacatctcag aaccagtcgc atggtgttga 174960 atccttctca tactcccatc tctttgacct tttgtcattg cacgtctctc tgtcaccatg 175020 tagcagggaa agcttcttga cttttaagtg cacagtcacc catgtgacta ggttgggctc 175080 acctggataa tccaggataa tcttcccaac tcaaggtcca taaccctcat cacatctgca 175140 aaatcccttt caccatgcga tacaacatat tcacagggat taaggcaagg gaatctttca 175200 gagacagttg ttttttcagc ctcgtctgaa atgtgctaag tgctttatat acatgtttaa 175260 agttatctcc attttgccag aagaaataaa gtcttagaaa aattgggcga attggctacc 175320 ttcctatgaa atgacagtca ttatttaaac ccatgtctac ttgatcctaa gtcatattgc 175380 tcttaattcc tataccatgc tgtgctgtgt gtatatccta ctatatatgt gcagcacaca 175440 cacacacacc ccaacatgtt atgaactgag tgttatttga aaaatcatct tgggccacat 175500 aaaatgtata gcttacaccg tttcttctag tactctttac accaattagt tttgtatatt 175560 agtaatctga gactcaggga aacgtggcat cttccaaact aatggcaagg ctacaaaaac 175620 agaaaatatt tattggccaa tttattagtt ttcctagact ttcaaaatgt agttatttca 175680 cagattagct atgaaactcc accttgatga tagcatacta aagaacatga agaaattaca 175740 ttgttttagt ttctaaaata gatttttaat ttctaagata ttagaatagt taaatagaaa 175800 tcacaaaata atatgcttat gttttatatg cttataattt gggcatagtt ttttgagttg 175860 acaattaagt tttagaattg cagtataaac agaagggaaa tgtgtgagtc ctttggccat 175920 tccctgttgt tgatcacaac acgtagaaac agctgcccca agctcttgta aatgtatctt 175980 tgccttagcc cttaactatg tttttttctg ttttagccat cctttttaat cccacttgtt 176040 tatcttttct taaaatgtac taactattca tagctgcaga tatttttctt aattctacta 176100 tcttagtttc attttctctg tggcacttgc tttacagata tctcagtgga gtgaaggatg 176160 tgctacatgt gctgcttcgc tgatctgccc actagactcc gatttgactc gttttcatat 176220 cttgctttcc cagagtctgc tgtcattgcg aaagcattgt ttattggtct ccctttcttc 176280 tttgctgcag aaccttgatg ctctgcacga ccaccttgct accatctacc caggaatgcg 176340 tgcaccttcc tttaggtgca ctgatgcaga aaagggcaaa ggactcattt tgcactacta 176400 ctcagagaga gaaggacttc aggatattgt cattggaatc atcaaaacag tggcacaaca 176460 aatccatggc actgaaatag acatgaaggt aacaaacagc aatggagact tctgaacaca 176520 gatgacatct aaaaatattt taaatgacac tctctaaatt tacctgcagt tatcactgtt 176580 agtgctcttc cctggaagta tatataagcc aaaacagtgg aaagaccagc aatcttctat 176640 atttggcctg ggagtgcatt atacaggata tttttttcct tctggcagca gatttttctt 176700 tctaattata tagtgttcaa ggctacaaga aatttacttt gcattccctg tgaacacaca 176760 ccagctggga agcagaagga tatgtgacga ctgagctgtg ggtttcagca tgcaaaatat 176820 ccataatatg gatagtcaaa ttctaaggca atataaaatt aagtattatt ttatagatca 176880 ttttctcttt gtcgattttt gtctgccctg aaatatagtt ggaatgtcta attttgtgaa 176940 tgaaagtgtt tgtttttgtt tttgttttta atcaagttac ttggtgaaag aagccaggtc 177000 tggtacttta aaaattgtgc tgttattgat aatacacact cttagttttg agtgatgatt 177060 tcaaggtact gatagggcaa ccgtacaaca aagagaacca ctgtttctct ttacctctgg 177120 cccttacggt ttctcccagt caggctgctt tttatgtgtt cagcatgttc atacccaaga 177180 gtcaattctc agtagcttta aacttgaggc caaagtgggg agatagatac ctttttccaa 177240 cctttctttg tttatagaaa acctattaga cagttctttc ttatagatca tagatatatt 177300 gtaacacact tgggaggtct aaaaccctta agtggagtat tatccaaaat taaaatagta 177360 acatttaaat aaattagaaa gatattccac atatttaatc tagtaaagtt atgttagagt 177420 tcatggtttt ctttctttca gaagttggtg ttagttctta cgtggcacct tttggtgatt 177480 tccaggtgca tcgttagttt tctacaacca ttagtaatgg cggaaaccac gattactgtt 177540 gcaccaacct aataga 177556 7 21 DNA Mus musculus 7 catgatgcga tcacaggagg c 21 8 21 DNA Mus musculus 8 cgcccggagc ctaggaagca g 21 9 21 DNA Mus musculus 9 ctctctgtgt gtgagagaga g 21 10 25 DNA Mus musculus 10 gtcagtgtca gacctgaaga tgctg 25 11 21 DNA Mus musculus 11 cccttccttg cttctcagta c 21 12 27 DNA Mus musculus 12 ctgctacaag cattgcctag acggacg 27 13 21 DNA Mus musculus 13 gacaccatgt acggtttcgt g 21 14 20 DNA Mus musculus 14 ctccaccttg tagacatcca 20 15 19 DNA Mus musculus 15 tgcacttcag agaaccttg 19 

What is claimed is:
 1. An isolated nucleic acid comprising a region having a nucleotide sequence that encodes the polypeptide sequences of SEQ ID NO:
 2. 2. The nucleic acid of claim 1, wherein the region is further defined as having the nucleotide sequence of the nucleotide sequence of SEQ ID NO:
 1. 3. An isolated and purified polynucleotide comprising a base sequence that is identical or complementary to a segment of at least 15 contiguous bases of SEQ ID NO:
 1. 4. The polynucleotide of claim 3, wherein said polynucleotide hybridizes to a polynucleotide that encodes a polypeptide comprising the amino acid residue sequence of SEQ ID NO: 1 or to the complement of such a sequence.
 5. The polynucleotide of claim 3 wherein said polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 20 contiguous bases of SEQ ID NO:
 1. 6. The polynucleotide of claim 3 wherein said polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 30 contiguous bases of SEQ ID NO:
 1. 7. The polynucleotide of claim 3 wherein said polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 50 contiguous bases of SEQ ID NO:
 1. 8. The polynucleotide of claim 3 wherein said polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 100 contiguous bases of SEQ ID NO:
 1. 9. The polynucleotide of claim 3, wherein said polynucleotide comprises a base sequence that is identical or complementary to all contiguous bases of SEQ ID NO:
 1. 10. An expression vector comprising a polynucleotide that encodes a polypeptide comprising an amino acid residue sequence of SEQ ID NO:
 2. 11. The expression vector of claim 11, wherein said polynucleotide comprises the nucleotide base sequence of SEQ ID NO:
 1. 12. The expression vector of claim 10, wherein said polynucleotide is operatively linked to an enhancer-promoter.
 13. A recombinant host cell comprising a polynucleotide that encodes a polypeptide comprising an amino acid residue sequence of SEQ ID NO:
 2. 14. The recombinant host cell of claim 13, comprising a polynucleotide that encodes a polypeptide comprising the amino acid residue sequence of SEQ ID NO:
 2. 15. The recombinant host cell of claim 14, wherein said polynucleotide comprises the nucleotide base sequence of SEQ ID NO:
 1. 16. The recombinant host cell by claim 13, wherein said polynucleotide is introduced into said cell by transformation of said cell with a vector comprising said polynucleotide.
 17. The recombinant host cell of claim 13, wherein said host cell expresses said polynucleotide to produce the polypeptide.
 18. A process for preparing a cell expressing a polypeptide comprising the steps of (a) transfecting a cell with a polynucleotide that encodes a polypeptide comprising an amino acid residue sequence of SEQ ID NO: 2 to produce a transformed host cell; and (b) maintaining the transformed host cell under biological conditions sufficient for expression of said polypeptide in the host cell.
 19. The method of claim 18, wherein the polynucleotide comprises a region having a nucleotide sequence of SEQ ID NO:
 1. 20. The process of claim 18, further defined as a process for preparing a cell expressing a polypeptide comprising the amino acid residue sequence of SEQ ID NO:
 2. 21. The process of claim 18, further comprising purifying an expressed polypetide having the amino acid sequence of SEQ ID NO: 2 from the transformed host cell.
 22. An isolated nucleic acid comprising a region having a nucleotide sequence of SEQ ID NO:
 3. 23. An isolated and purified polynucleotide comprising a base sequence that is identical or complementary to a segment of at least 15 contiguous bases of SEQ ID NO:
 3. 24. The polynucleotide of claim 23 wherein said polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 20 contiguous bases of SEQ ID NO:
 3. 25. The polynucleotide of claim 23 wherein said polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 50 contiguous bases of SEQ ID NO:
 3. 26. The polynucleotide of claim 23 wherein said polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 100 contiguous bases of SEQ ID NO:
 3. 27. The polynucleotide of claim 23, wherein said polynucleotide comprises a base sequence that is identical or complementary to all contiguous bases of SEQ ID NO:
 3. 28. An expression vector comprising a polynucleotide that comprises the nucleotide base sequence of SEQ ID NO:
 3. 29. The expression vector of claim 28, wherein said polynucleotide is operatively linked to an enhancer-promoter.
 30. A recombinant host cell comprising a polynucleotide that comprises the nucleotide base sequence of SEQ ID NO:
 3. 31. An isolated nucleic acid molecule comprising a region having a nucleic acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or a fragment thereof, the region further defined as encoding a murine alpha1 soluble guanylyl cyclase possessing a genomic organization as shown in Table
 6. 32. An expression vector comprising a nucleic acid having region having a sequence of, or complementary to, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a portion of any of these.
 33. The expression vector of claim 32, wherein said polynucleotide is operatively linked to an enhancer-promoter.
 34. A recombinant host cell comprising nucleic acid having region having a sequence of, or complementary to, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a portion of any of these.
 35. The recombinant host cell of claim 34, wherein said polynucleotide comprises the nucleotide base sequence of SEQ ID NO:
 1. 36. The recombinant host cell by claim 34, wherein said polynucleotide is introduced into said cell by transformation of said cell with a vector comprising said polynucleotide.
 37. The recombinant host cell of claim 34, wherein said host cell expresses said polynucleotide to produce the polypeptide.
 38. A process for preparing a cell expressing a polypeptide comprising the steps of (a) transfecting a cell with a nucleic acid having region having a sequence of, or complementary to, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a portion of any of theseto produce a transformed host cell; and (b) maintaining the transformed host cell under biological conditions sufficient for expression of said polypeptide in the host cell.
 39. The method of claim 38, wherein the polynucleotide comprises a region having a nucleotide sequence of SEQ ID NO:
 1. 40. The process of claim 38, further defined as a process for preparing a cell expressing a polypeptide comprising the amino acid residue sequence of SEQ ID NO:
 2. 41. The process of claim 38, further comprising purifying an expressed polypetide from the transformed host cell.
 42. The process of claim 38, further defined a process of producing an active enzyme.
 43. A method for the detection of genetic and/or inherited and /or acquired human diseases utilizing a nucleic acid comprising a region having a sequence of, or complementary to, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a portion of any of these.
 44. A diagnostic kit for the detection of genetic and/or inherited and /or acquired human diseases utilizing a nucleic acid comprising a region having a sequence of, or complementary to, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a portion of any of these.
 45. A method of treating disease comprising utilizing a nucleic acid comprising a region having a sequence of, or complementary to, SEQ ID NO: 1, SEQ ID NO: 3, SEQ 20 ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a portion of any of these.
 46. A method of screening for drugs, drug design, and/or drug development comprising utilizing a nucleic acid comprising a region having a sequence of, or complementary to, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a portion of any of these. 